Bacteria contain DNA polymerase I (PolI), a single polypeptide chain consisting of ∼930 residues, possessing DNA-dependent DNA polymerase, 3′-5′ proofreading and 5′-3′ exonuclease  activities. PolI is particularly important in the processing of Okazaki fragments generated during lagging strand replication and must ultimately produce a double-stranded substrate with a nick suitable for DNA ligase to seal. PolI's activities must be highly coordinated both temporally and spatially otherwise uncontrolled 5′-nuclease activity could attack a nick and produce extended gaps leading to potentially lethal double-strand breaks. To investigate the mechanism of how PolI efficiently produces these nicks, we present theoretical studies on the dynamics of two possible scenarios or models. In one the flap DNA substrate can transit from the polymerase active site to the 5′-nuclease active site, with the relative position of the two active sites being kept fixed; while the other is that the 5′-nuclease domain can transit from the inactive mode, with the 5′-nuclease active site distant from the cleavage site on the DNA substrate, to the active mode, where the active site and substrate cleavage site are juxtaposed. The theoretical results based on the former scenario are inconsistent with the available experimental data that indicated that the majority of 5′-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus. By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data. We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5′-3′ exonuclease activities. Moreover, predicted results for the latter model are presented. DNA polymerase I (PolI) is a well-characterized enzyme involved in DNA replication and repair in vitro DNA synthesis on circular single-stranded DNA or at a nick within a duplex DNA substrate These enzymes have the ability to degrade flap structures, single-stranded and double-stranded DNA from free 5′ ends  [9]. Thein vivo replication of the lagging strand DNA E. coli. Although the 5′-3′ exonuclease activity of PolI has long been known to be essential for viability in Streptococcus pneumoniaeE. coli, Bacillus subtilis and Synechococcus elongatesPolI carries out similar processing of Okazaki fragments during MycoplasmaEukaryotes possess multiple homologues of the PolI 5′-3′ exonuclease domain et al. The consequences of inappropriate Okazaki fragment processing could be catastrophic to the cell . Should To perform its biological function, the physically linked polymerase and 5′-nuclease domains of PolI see  must colIt is hypothesized that the 5′-nuclease domain has a high affinity for the flap DNA substrate having no single-stranded gap , which ixyz as shown in y axis is perpendicular to the paper surface. The interaction potential of the DNA substrate with the polymerase domain and the 5′-nuclease domain can be written asU0 is the interaction strength of the DNA substrate with the polymerase domain or the 5′-nuclease domain. U1(x), U2(y) and U3(z) can be written in the following formsx, y, z)  =   while the 5′-nuclease active site is positioned at   =  . The parameter A characterizes the interaction distance of the potential. For clarity, the forms of U , U  and U  are shown in U gave only slightly different statistical results but has no effect on our conclusion (see below).Now, based on the model we carried out a quantitative study of the dynamics of DNA transition. Take the coordinate Oγ is the frictional drag coefficient on the DNA and i =  x,y,z) is the fluctuating Langevin force with Dr = 3 nm in a solution with viscosity of η = 0.01 gThe movement of the DNA substrate relative to the PolI in viscous solution can be described by Langevin equations:A = 0.5 nm as is consistent with the Debye length in the order of 1 nm in solution   =   to the 5′-nuclease active site at   =  , while nP, for the DNA to transfer to the 5′-nuclease active site versus the interaction strength U0 are shown in nP. The statistical results of the mean time, dT, for the DNA to detach from the polymerase   =   versus U0 are shown in U such as that plotted in To study the transition dynamics of the flap DNA substrate, we solved Eqs. (8)–(10) numerically by using the stochastic Runge-Kutta method tion see . In FigunP increases only slowly with the increase of U0 while dT increases significantly with the increase of U0. When U0>11 kBT, dT increases exponentially with U0. From U0 = 20 kBT, the probability nP that the DNA substrate transfers from the polymerase active site to the 5′-nuclease active site of the same PolI is only slightly larger than 0.1, whereas the probability for the DNA to dissociate into solution is nearly 0.9. This implies that only a minority of 5′-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus. In other words, in order to cleave the flap, the DNA substrate has to first dissociate from the polymerase domain of the PolI molecule that has just synthesized the flap and then bind to the 5′-nuclease domain of another PolI molecule. This is inconsistent with the experimental data that indicate the majority of 5′-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus From Thus, our results do not support the model in which PolI performs the two cooperative activities via transition of the flap DNA substrate from the polymerase domain to the 5′-nuclease domain. In other words, the rigid PolI model is an unreasonable one to describe the cooperation of the polymerase and 5′-nuclease domains of PolI so as to leave a nick in the flap DNA substrate.In above section, we showed that the rigid PolI model is an unreasonable one to describe the cooperation of PolI's polymerase and 5′-nuclease activities. In this section we examine the feasibility of the flexible PolI model.N = 16 amino acids It is known that the polymerase domain and the 5′-nuclease domain are connected by a linker of Based on the above hypotheses, the model for coordination between polymerase and 5′-nuclease activities via transition of the 5′-nuclease domain from the inactive to active modes is presented as follows. We begin with the polymerase active site positioned at the replication fork, with the downstream 5′-flap being very far away from the 5′-nuclease domain . In thisa is the average size of an amino acid. With BkT = 4.11 pNT = 298 K), N = 16 and a = 0.3 nm, we obtain from Eq. (11) that K = 8.56 pN/nm, which is in good agreement with the experimental value of about 8.5 pN/nm K is varied, we take K = 8.56 pN/nm. The good agreement between the calculated and experimental values of K implies that the connection between the 5′-nuclease domain and the polymerase domain is only via the flexible linker and other types of the interaction between the two domains such as electrostatic force, van der Waals force, etc., can be negligible.First, we determine the magnitude of the spring constant resulting from “entropic” spring of the linker coil. The spring constant can be simply calculated by r = 3.5 nm , where the coordinate Oxy is shown in xy plane here to be the same as that shown in z direction that is perpendicular to the paper surface in x, y) while the rotation by its rotation angle θ. At equilibrium position of the 5′-nuclease domain relative to the polymerase domain, i.e., when the linker is not stretched, the center-of-mass position and rotation angle of the 5′-nuclease domain is written as   =    and the domain is rotated counterclockwise by an angle of θ, the linker is stretched by a length of R =  x and y directions are x, y, θ)  =  , as schematically seen in d should be in the range of 0<d<3 nm. Here, we take d as a variable parameter in our calculations. Similar to Eq. (1), the interaction potential of the 5′-nuclease domain with the flap DNA substrate can be written as followsV0 is the interaction strength. V1(x), V2(y) and V3(θ) have the following formsrB = A = 0.5 nm.To simplify our analysis of the effect of the elastic force of the stretched linker on the movement and rotation of the 5′-nuclease domain, we approximate the 5′-nuclease domain as a sphere of radius 5 nm see . Here, wr, 0, 0) . When th =   see . Thus, tNa  = 4.8 nm; 2i =  x,y,θ) satisfies i ≠ j) and x, y,θ)  =   and   =  , respectively.Thus, the movement and rotation of the 5′-nuclease domain in viscous solution can be described by the following Langevin equationsx, y,θ)  =   to the final state   =   is insensitive to the value of the interaction strength V0 between the 5′ nuclease domain and the flap DNA substrate (We solved Eqs. (17)–(19) numerically by using the stochastic Runge-Kutta method, as mentioned above. The calculated results show that the mean time for the 5′-nuclease domain to transit from the inactive to active modes, i.e., the mean time for the 5′-nuclease domain to transit from the initial state . It is seen that mT increases with the increase of d when d>2.5 nm. Even for the large value of d, e.g., d = 3 nm, mT is only about 10 µs, which is a very short time. On the other hand, from U0 is only larger than 13 BTk, the mean dissociation time of the DNA from the polymerase is dT>10 µs. In other words, if U0 is only larger than 13 BkT, the 5′-nuclease domain has a large probability to bind the flap DNA strongly before its dissociating from the polymerase domain. The affinity of 13 BkT corresponds to a mean dissociation constant larger than 1 µM, which is a very weak affinity between proteins and DNA. Available experimental data indicated that the polymerase domain binds dsDNA . To see the effect of the variation of temperature on the results, we change the temperature in our calculations. The value of viscosity data see . The calloadF, acting on the residues indicated by the blue dot , (18) and (19) should be added by terms mT versus loadF at T = 298 K (25°C) are shown in mT increases significantly with the increase of the external load. It is interesting to note that, only at loadF>2.5 pN, mT>600 µs that is much larger than dT = 133 µs at U0 = 16 BkT (see above). This implies that the flap DNA substrate becomes dissociated from the polymerase before the 5′-nuclease domain transits to the active mode. Thus, the polymerase molecule that has just extended the upstream primer terminus cannot perform the activity of cleaving the downstream 5′-flap, i.e., the polymerase cannot simultaneously perform the two activities.Next, we present our predicted results for the effect of the external force on the transition of the 5′-nuclease domain from the inactive to active modes. Consider a load, blue dot  along thIn this work, our theoretical results indicate that, to perform the 5′-nuclease activity, it is reasonable that the 5′-nuclease domain transits from its equilibrium position to the position near the polymerase active site (the flexible PolI model) rather than the flap DNA substrate transits from the polymerase active site to the 5′-nuclease active site (the rigid PolI model). By contrast, during the proofreading process, it is reasonable that the DNA transits between the polymerase active site and the 3′ – 5′ exonuclease active site. In detail, in order to cleave the mismatched base, the 3′ – 5′ single-stranded DNA is argued to transit from the polymerase active site to the exonuclease active site by unwinding some base pairs Taq polymerase bound to the inhibitory Fab observed by Urs et al. et al. Taq polymerase bound to the inhibitory Fab With the flexible PolI model, the difference in the position of the 5′-nuclease domain relative to the polymerase domain in the crystal structure of in vivo. It is also of particular importance in the widely used Taqman, or fluorogenic 5′ nuclease assay Taq polymerase and is detected by an increase in fluorescence that occurs on separation of the reporter and quencher dyes. This leads to loss of Forster resonance energy transfer (FRET) between the donor and quencher dyes and an increase in signal at the emission wavelength of the fluorophore. When a fragment containing this target DNA is subjected to PCR amplification using Taq polymerase with flanking primers, the target probe is cleaved by the 5′ nuclease activity provided the probe is fully annealed to its target. If a mismatch is present within the otherwise complementary sequence, the polymerase activity simply displaces the probe rather than hydrolyzing it. Thus, understanding the mechanisms of 5′ nuclease/polymerase coordination may be useful in designing more efficient 5′ nuclease assays.The balanced action of DNA PolI's 5′ nuclease and polymerase activities is not only important loadF>2.5 pN acts on the 5′-nuclease domain, the polymerase molecule that has just extended the upstream primer terminus cannot perform the activity of cleaving the downstream 5′-flap , palm (grey), fingers (green), proofreading domain (purple), and 5′ -nuclease domain (dark blue) are shown as a backbone cartoon rendered using Pymol (DeLano Scientific). An oversized grey sphere marks the active site of the 5′-nuclease domain, while red spheres provide reference to the position occupied by dCTP in the active site of the polymerase (modeled from 5KTQ.pdb). The distance between these two features is indicated in Å.Experimentally observed x-ray structure of (TIF)Click here for additional data file.Figure S2U , U  and U , with A = 0.5 nm and U0 = kBT.Forms of interaction potential of the polymerase domain and the 5′-nuclease domain with the flap DNA substrate, (TIF)Click here for additional data file.Figure S3U0 = 16 kBT.A typical result for the trace of DNA relative to the DNA polymerase. (TIF)Click here for additional data file.Figure S4U0 = 16 kBT.A typical result for the trace of DNA relative to the DNA polymerase. (TIF)Click here for additional data file.Figure S5U0 = 16 kBT.A typical result for the trace of DNA relative to the DNA polymerase. (TIF)Click here for additional data file.Figure S6U0 = 16 kBT.A typical result for the trace of DNA relative to the DNA polymerase. (TIF)Click here for additional data file.Figure S7U , U  and U  being shown in (A), (B) and (C), respectively. U0 = kBT.Another form of the interaction potential of the polymerase domain and the 5′-nuclease domain with the flap DNA substrate, with (TIF)Click here for additional data file.Figure S8mT versus the spring constant K for different values of d. Dotted line corresponds to K = 8.56 pN/nm. V0 = 18 kBTCalculated results of the mean transition time (TIF)Click here for additional data file.Figure S9mT for the 5′-nuclease domain to transit from the inactive to active modes as a function of the interaction strength V0 between the 5′-nuclease domain and the flap DNA substrate, with d = 2 nm.Calculated results of the mean time (TIF)Click here for additional data file.Figure S10d = 2 nm. (B) d = 2.5 nm.Time distributions of the 5′-nuclease domain transiting from the inactive to active modes. (A) (TIF)Click here for additional data file.Figure S11loadF acting on the residues (blue dots) of the 5′-nuclease domain. (A) Equilibrium position of the 5′-nuclease domain relative to the polymerase domain. (B) A transient position of the 5′-nuclease domain.Schematic diagram to illustrate the external load (TIF)Click here for additional data file.Table S1Temperature dependence of viscosity.(DOC)Click here for additional data file.


Signal transduction pathways involving the second messenger cyclic di-GMP [bis-(3′-5′)-cyclic di-guanosine monophosphate] occur widely in bacteria where they act to link perception of environmental or intracellular cues and signals to specific alterations in cellular function. Such alterations can contribute to bacterial lifestyle transitions including biofilm formation and virulence. The cellular levels of the nucleotide are controlled through the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). The GGDEF domain of DGCs catalyses the synthesis of cyclic di-GMP from GTP, whereas EAL or HD-GYP domains in different classes of PDE catalyse cyclic di-GMP degradation to pGpG and GMP. We are now beginning to understand how alterations in cyclic di-GMP exert a regulatory action through binding to diverse receptors or effectors that include a small ‘adaptor’ protein domain called PilZ, transcription factors and riboswitches. The regulatory action of enzymically active cyclic di-GMP signalling proteins is, however, not restricted to an influence on the level of nucleotide. Here, I will discuss our recent findings that highlight the role that protein–protein interactions involving these signalling proteins have in regulating functions that contribute to bacterial virulence. Acetobacter xylinum (now Gluconacetobacter xylinus) (Cyclic di-GMP [bis-(3′-5′)-cyclic di-guanosine monophosphate] was originally described in 1987 as an allosteric regulator of cellulose synthesis in xylinus)  (Ross etxylinus)  , a two-component system comprising the sensor kinase RpfC and HD-GYP domain regulator RpfG is implicated in perception and signal transduction of the cell-to-cell signal DSF  (In the plant pathogen  factor) . This Rp factor) . Our stuXcc and their broader relevance to an understanding of regulation of virulence by cyclic di-GMP in other pathogens are discussed. This begins with a brief overview of the DSF signalling system in Xcc, before going on to review in more detail what is known of the multiple regulatory functions of RpfG and the HD-GYP domain, how such diverse regulatory actions are exerted and the bifunctional nature of certain GGDEF domain proteins.Herein my group’s findings from studies of the Rpf/DSF system in rpf gene cluster (for regulation of pathogenicity factors) was identified originally as being required for full virulence in Xcc (rpfA–I) that are located within a 21.9 kb region of the chromosome of Xcc. Mutation of rpfF, rpfG and rpfC genes leads to a coordinate downregulation of the synthesis of a number of extracellular enzymes, including endoglucanase, protease, endomannanase and polygalacturonate lyase, and of the extracellular polysaccharide xanthan, but an increase in biofilm formation    to the expression of genes encoding extracellular enzymes  binding domain and a C-terminal DNA-binding domain  of those whose transcript level is significantly altered. Contrary to expectation, the regulatory effects of RpfC and RpfG on transcript levels have limited overlap. Furthermore, mutations of rpfG and rpfC have divergent effects on the transcript levels of a number of genes  analysis  leads to a reduction in motility, but has no effect on the synthesis of extracellular enzymes or biofilm formation.The strategy to examine the biological role of HD-GYP : : GGDEF domain interactions in  XC_0420 . AlanineXcc. The DxD motif is distinct from the catalytic A site (GGD/EEF) or the regulatory I site and is conserved in a number of GGDEF domain proteins, suggesting that GGDEF : : HD-GYP interactions may be similarly widespread.A parallel approach using synthetic peptide overlay technology (SPOT) in conjunction with alanine-substitution mutagenesis allowed the identification of the DxD motif within the GGDEF domain proteins as being required for inter-domain interaction  as bait was used to identify possible targets for XC_2249 (PilZ) action .Xcc, an effect that is likely to be exerted through an influence on distinct and multiple functions.As detailed above, DSF signalling promotes the formation of a complex between the HD-GYP domain response regulator RpfG and two GGDEF domain proteins to regulate pilus-dependent motility . The intCaulobacter crescentus requires the response regulator CpdR, while the GGDEF domain protein PopA, which lacks DGC activity, recruits the cell cycle regulator CtrA to the pole  with an HD-GYP domain and a third protein, PA2572, which contains a variant domain with the sequence YNIGK in place of HDMGK seen in RpfG  and only two PDEs; PdeA has an EAL domain and PdeB has an HD-GYP domain. Mutational analysis indicates that Rrp1 is essential for survival of the spirochaete in the tick vector, Ixodes scapularis, but not for infection of mice, whereas PdeA is required for infectivity in mice. Mutation of pdeB leads to a reduced ability to survive in the tick vector and significantly increases flexing, indicating a role for this HD-GYP domain protein in motility. Furthermore, the in vitro activity of the recombinant HD-GYP domain protein PdeB as a PDE against cyclic di-GMP has been demonstrated.A number of recent studies have described elements of cyclic di-GMP signalling in the Lyme disease spirochaete, pathogen . The B. V. cholerae has addressed the role that HD-GYP domain proteins may have in signal transduction mechanisms associated with quorum sensing (QS) in the El Tor biotype  that encode HD-GYP domain proteins in V. cholerae El Tor is activated by QS. Each of the encoded proteins has the conserved HD and GYP motifs, as well as several other conserved residues, suggesting that they are active as PDEs. In V. cholerae El Tor, QS negatively regulates the production of exopolysaccharide and biofilm formation. Accordingly, overexpression of VCA0681 in V. cholerae El Tor leads to a reduction in cellular levels of cyclic di-GMP, a decrease in expression of the vps gene cluster which directs extracellular polysaccharide synthesis and a reduction in biofilm formation. These regulatory effects are associated with the PDE activity of VCA0681; a variant with a D to A substitution within the HD catalytic diad has little effect on cyclic di-GMP levels and does not influence either vps expression or biofilm formation. In contrast to overexpression, mutation of individual genes encoding HD-GYP domain proteins does not lead to any alteration in vps expression or biofilm phenotype, suggesting possible redundancy in the action of these proteins.Work in  biotype . Using lentified . It was The findings from these three studies illustrate two aspects of HD-GYP domain proteins that are consonant with our understanding of the diverse roles of other cyclic di-GMP signalling proteins. The first is that different HD-GYP domain proteins appear to have distinct regulatory roles. The second is that although domains with sequences that diverge from the consensus may have lost enzymic activity, they may still play a regulatory role.Xcc has improved our knowledge of an element of the ‘core pathway’ of cyclic di-GMP signalling in bacteria, we still have limited understanding of the full role of this domain and the diversity of its action in different bacteria. Although studies of RpfG have revealed some insight into the role of signature  residues, the role of other highly conserved residues, such as those within the more extended motif HHExxDGxGYP, is unknown. These residues could conceivably have roles in metal co-factor and substrate binding, enzymic action or in protein–protein interactions with GGDEF domain proteins. High-resolution crystallography has already provided great insights into the mechanisms of action of GGDEF and EAL domain proteins and various cyclic di-GMP effectors (Although identification and characterization of the role of the HD-GYP domain in the regulation of bacterial processes in ffectors . HoweverAlthough the interaction between RpfG and two GGDEF domain proteins is known to regulate motility, it is not known how protein–protein interactions between the HD-GYP : : GGDEF : : PilZ complex affect the activities of PilT and PilU, and whether they have influences on other bacterial functions. Equally, it is not known whether interaction with other GGDEF domain proteins occurs under different environmental or growth conditions, and whether these interactions serve to regulate different bacterial functions or processes. In addition, the biological relevance of HD-GYP domain interaction with other classes of bacterial regulatory protein that have been revealed by several Y2H analyses should be assessed.Xcc but also more widely in other pathogenic bacteria. Since HD-GYP domain proteins are known to regulate expression of virulence determinants or biofilm formation in several of these pathogens, elucidation of these pathways may identify key steps for interference with consequences for novel strategies for disease control.The results of work that addresses these issues should contribute to our understanding of signal transduction pathways that involve the HD-GYP domain and cyclic di-GMP, not only in


The unfolded protein response (UPR) is a cell-signaling system that detects the accumulation of unfolded protein within the endoplasmic reticulum (ER) and initiates a number of cellular responses to restore ER homeostasis. The presence of unfolded protein is detected by the ER-luminal sensor domains of the three UPR-transducer proteins IRE1, PERK, and ATF6, which then propagate the signal to the cytosol. In this review, we discuss the various mechanisms of action that have been proposed on how the sensor domains detect the presence of unfolded protein to activate downstream UPR signaling. Eukaryotic secretory and membrane proteins attain their functional and assembled conformation in the endoplasmic reticulum (ER) prior to transit to the Golgi apparatus. A number of molecular chaperones, folding enzymes and co-factors assist in the folding, post-translational modification, and trafficking of the newly translated polypeptide chains. Protein quality control mechanisms, such as the calnexin/calreticulin chaperone system, ensure only properly folded proteins leave the ER . If protSaccharomyces cerevisiae where the response to ER stress was found to be mediated uniquely by the inositol requiring enzyme 1 (IRE1) protein (referred to as yIRE1 in this review) [The UPR was first characterized in  review) . In mamm review) . Each of review) . This li review) . These g review) –9. IRE1  review) ,11. RIDD review) . If the  review) .The luminal senor domains of the three UPR transducers detect the accumulation of unfolded protein within the ER . The senet al. and Kohno et al.[The most supported mechanism by which ER-stress initiates the UPR is the “competition model” by which BiP binds to the ER luminal sensor domains of IRE1, PERK and ATF6 in unstressed cells keeping them in an inactive monomeric state. Upon ER-stress, the high concentrations of unfolded proteins compete for BiP binding, thereby sequestering it from the luminal sensor domains. This action allows the sensor domains to dimerize/oligomerize with subsequent propagation of the signal via activation of their cytosolic domains ,13 see . Correlano et al.,15. Earlno et al.,17. Furtno et al.,19. DissFrom the studies above, however, it cannot be excluded that senor domain binding to BiP is actually only a client interaction and the decrease in UPR signaling, when BiP is over-expressed, is due to a reduction in levels of unfolded proteins rather than repression of UPR ,21. OverBiP binding was initially thought to occur in the dimerization region of the sensor domains, thereby keeping the senor domain in an inactive state by steric occlusion. BiP release would unmask the dimerization interface allowing dimerization to occur, thus acting as a negative regulator and giving rise to the competition model of activation. This model of repression was also suggested for ATF6, where its ER-stress responsive region contains two Golgi-localization sequences. When bound with BiP, these regions are masked and ATF6 is retained in the ER .et al. show that deletion of region V results in the loss of BiP-binding and that such deletion mutants remarkably have no effect upon UPR signaling and they actually retain their ability to be fully inducible upon ER-stress [The exact model of BiP binding and dissociation from the UPR sensors and how this leads to their activation is not yet understood. In an attempt to identify the BiP binding region, a series of scanning deletion mutants were generated and analyzed for their ability to activate the UPR ,22,23. TR-stress . These sR-stress ,22,25,26R-stress .Hsp70 chaperones consist of a nucleotide-exchange domain and a substrate-binding domain, their ATPase activity being key for the control of substrate binding and release. The ATPase activity of BiP may also be important in regulating its binding and release from the luminal sensor domains, thus impacting on UPR activation. In the ATP-bound form, Hsp70s, have an “open” low substrate-affinity conformation, whereas in the ADP-bound form, the substrate-binding domain switches to a “closed” high substrate-affinity conformation, thus trapping the bound substrate. The ATPase activity of Hsp70s is known to be stimulated upon substrate association changing them from an “open” low substrate affinity ATP-bound conformation to a “closed” high substrate affinity ADP-bound form.Targeted temperature sensitive (ts) mutations present within the ATPase domain lock BiP in “open” ATP-bound state blocking activation of the UPR whilst still associating with IRE1, whereas ts mutations in the substrate-binding domain not only abrogate interactions with unfolded protein substrate but also lead to constitutive activation of the UPR and loss of association with IRE1 . This suIn contrast, ATF6 binding to BiP does not seem to depend on nucleotide cycling as their association was not dependent on the addition of specific nucleotides . BiP mutin vitro. Interestingly, two further studies have shown direct binding of yIRE1 luminal domain to unfolded proteins and this was a prerequisite for its oligomerization and activation [in vitro and to activation [An alternative “BiP-independent” model of UPR activation, which points towards a direct role for unfolded proteins in UPR activation, has been proposed, at least in the yeast system. The 3.0 Å crystal structure of the yIRE1 core luminal domain (residues 114–449) revealed the presence of a groove located at the dimerization interface, interface 1 (IF1), which resembles the peptide-binding domains of major histocompatibility complexes (MHCs) . In its tivation ,34. To fin vivo, highlighting the biological relevance of this process at least in the yeast system [The crystal lattice in the yIRE1 luminal domain crystals revealed the presence of a secondary interaction site: interface 2 (IF2), which may allow for higher oligomer formation in a putative helical arrangement. A mutation at IF2, W426A, also decreased activation of the UPR element (UPRE) reporter, thus suggesting that the clustering of yIRE1 luminal domains via IF2 is involved in the initiation of downstream signaling. Several mutations that compromised yIRE1 cluster formation were shown to disrupt UPR signaling t system ,35. Gardt system . In a met system .et al. argue that the MHC-like groove is too narrow and structurally unfavorable to accommodate peptide binding. Firstly, the conserved G105 residue in IRE1α forms a hydrogen bond to contribute to dimer stability, and in doing so, blocks access to the proposed peptide-binding groove. Secondly, two of the residues corresponding to those speculated to contribute to peptide binding in yIRE1, T161 and T179, are either buried or not conserved in the IRE1α groove. Finally, projection of IRE1α cytosolic domain suggests that the MHC-like groove faces the ER membrane thus it is unlikely that protein binding would occur. However, IRE1α was also found to form clusters on the ER membrane of human cells upon induction of ER-stress and this correlated with its phosphorylation and activation of the RNase domain [Based on the crystal structure of IRE1α luminal domain it was suggested that the direct binding of unfolded proteins is neither probable nor necessary for initiation of the UPR . Zhou ete domain . Anothere domain . Based oe domain . In addiet al. showed that yIRE1 mutants that are unable to form clusters have impaired ability to activate the UPRE reporter, whereas mutants that constitutively cluster still required extrinsic ER-stress to achieve full activation [et al. were able to uncouple the two downstream signaling branches in vitro[An alternative two-step mechanism has been suggested in which the release of BiP acts as prerequisite to unfolded protein binding to the senor domains . Kimata tivation . Togethetivation . From thtivation . Upon petivation ,40. Thistivation . By usin in vitro. It is iet al. showed that the cytosolic domain of yIRE1 can sense ER membrane distortions leading to activation of downstream pathways independently of ER-stress [Recent studies have suggested that IRE1 activation at least can be modulated by extrinsic factors, even those coming from the cytosol. For example, the flavonol quercetin has been shown to dimerize and weakly activate yIRE1 even in the absence of ER-stress. The co-crystal structure revealed quercetin binds to a novel ligand binding pocket of yIRE1’s kinase domain and as such promotes dimerization of yIRE1’s cytosolic domain . SeveralR-stress . Once agFinally, two new and very different mechanisms involved in IRE1α and PERK, and ATF6 activation have recently been proposed. Firstly, Jwa and Chang recently suggested a role of PARP proteins in activation of IRE1α and PERK . PARP pret al. show that glycosylation in the luminal domain of ATF6 serves as a sensor for ER homeostasis and directs ATF6 signaling [N-glycosylation sites; however, these have not been linked to ER-stress sensing and their role remains unknown.Furthermore, Hong ignaling . The undignaling . Since gignaling . However


The concept of reticular chemistry is investigated to explore the applicability of the formation of Covalent Organic Frameworks (COFs) from their defined individual building blocks. Thus, we have designed, optimized and investigated a set of reported and hypothetical 2D COFs using Density Functional Theory (DFT) and the related Density Functional based tight-binding (DFTB) method. Linear, trigonal and hexagonal building blocks have been selected for designing hexagonal COF layers. High-symmetry AA and AB stackings are considered, as well as low-symmetry serrated and inclined stackings of the layers. The latter ones are only slightly modified compared to the high-symmetry forms, but show higher energetic stability. Experimental XRD patterns found in literature also support stackings with highest formation energies. All stacking forms vary in their interlayer separations and band gaps; however, their electronic densities of states (DOS) are similar and not significantly different from that of a monolayer. The band gaps are found to be in the range of 1.7–4.0 eV. COFs built of building blocks with a greater number of aromatic rings have smaller band gaps. In the past decade, considerable research efforts have been expended on nanoporous materials due to their excellent properties for many applications, such as gas storage and sieving, catalysis, selectivity, sensoring and filtration . In 1994One of the first families of materials synthesized using reticular chemistry were the so-called Metal-Organic Frameworks (MOFs) . They arIn 2005, Coté and co-workers introduced COF materials –14, wherCOFs, compared with MOFs, have lower mass densities due to the absence of heavy atoms and therefore might be better for many applications. For example, the gravimetric uptake of gases is almost twice as large as that of MOFs with comparable surface areas –16. BecaRecently, we have studied the structures of experimentally well-known 2D COFs . We haveIn the present work, we aim to explore how far the concept of reticular chemistry is applicable to the individual building units, which define COFs. For this purpose, we have investigated a set of reported and hypothetical 2D COFs theoretically by exploring their structural, energetic and electronic properties. We have compared the properties of the isolated building blocks with those of the extended crystal structures, and have found that the properties of the building units are indeed maintained after their assembly to a network.I–IV) and five linkers (a–e) for the systematic design of a number of 2D COFs (I and III (no linker is present): REF-I and REF-III. Properties of other COFs were compared with the properties of these two structures. Some of the studied COFs are already well known in the literature [8We have considered four connectors  and hexagonal [H] (connector IV), and the linkers between linear [l]  and trigonal [t] . Topology of the layer is determined by the geometries of the connector and linker molecules, and typically a hexagonal pattern is formed due to the D3h symmetry of the connector moieties. Based on these topologies of the constituent building blocks, we have classified the studied COFs into four groups: Tl, Tt, Hl and Ht (Using the secondary building unit (SBU) approach, we can distinguish the connectors between trigonal -4,4'-diylboronic acid (BPDA), 5’-(4-boronophenyl)--4,4”-diboronic acid (BTPA), benzene-1,3,5-triyltriboronic acid (BTBA) and pyrene-2,7-diylboronic acid (PDBA) were considered as linkers xtension . Self-coII, III and IV, respectively, and the corresponding linkers. Self-condensation of tetrahydroxydiborane (THDB) and co-condensation of HHB with THDB result in the formation of the reference structures, REF-I and REF-III, respectively. In relation to the corresponding connector/linker topologies, these building blocks satisfy the following equations of the co-condensation reaction for COF formation:Co-condensation of the above molecular units with compounds such as 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP), hexahydroxybenzene (HHB) and dodecahydroxycoronene (DHC)  gives ri−1 per formula unit.with a stochiometry appropriate for one unit cell. The number of covalent bonds formed between the building blocks is equivalent to the number of released water molecules, we refer to it as “formula unit” and will give all energies in the following in kJ·molWe have calculated the condensation energy of a single COF layer formed from monomers  according to the above reactions as follows:mtot is the total energy of the monolayer; EH2Otot is the total energy of the water molecule; Ebb1tot and Ebb2tot are the total energies of interacting building blocks; and n, m1, m2 are the corresponding stoichiometry numbers.where Es,b, of layers:We have also calculated the stacking energy, Enltot is the total energy of ns number of layers stacked in a COF. Finally, the COF formation energy can be given as a sum of Ec,b and Es,b. The calculated energies are given in where EI and II, while exothermic for the others, except for COF-5M, COF-6M and TP COF-2M. However, all reactions leading to COFs in their bulk forms are exothermic. It is to be noted that COF-1 is the least stable in AB form, which is in contrast with experimental observations [I and linker d is more stable in AB stacking, compared with other stacking kinds. Two other linker-d made COFs (COF-11M and COF-7M) have AB stacking energies comparable to other stacking forms. For all other COFs, AB stacking is disfavoured.All energies are given in terms of reaction energies, that is, more negative values indicate more stable products. Interestingly, the formation of monolayers is endothermic for the COFs made from connectors rvations , howeverrvations . NeverthAA stacking is always energetically disfavoured compared to the structurally close serrated and inclined stacking kinds. This is in contrary to experimental observations for most of the COFs. However, simple rationalisation, in terms of Coulomb energy as well as our XRD simulations presented in this and earlier work , supportc,b energies are 7.71, 1.49 and 0.40 kJ•mol−1, respectively; hence supporting the endothermic nature of the condensation reaction and is in reasonable agreement with our DFTB results  to support our results quantitatively. For simplicity we have used a finite structure instead of a bulk crystal. Their calculated E results .IV. The band gaps are different for different stacking kinds. This difference can be attributed to the different optimized interlayer distances. Generally, AB, serrated and inclined stacked COFs have band gaps comparable to, or larger than, that of their AA stacked analogues.All COFs, including the reference structures, are semiconductors with their band gaps lying between 1.7 eV and 4.0 eV . The larWe have calculated the electronic total density of states (TDOS) and the individual atomic contributions . The energy state distributions of COFs and their monolayers are studied and a comparison for COF-5 is shown in I connectors and different linkers are plotted in I, a structure which is composed solely of connector building blocks. Going from top to bottom of the plots, the number of carbon atoms per unit cell increases. It can be seen that this causes a decrease of the band gap. a linkers and different connectors, where the COFs are arranged in the increasing number of carbon atoms in their unit cells from top to the bottom. Again, the C PDOS is compared with that of graphite, while both REF-I and REF- III are taken in comparison to O and B PDOS. The observed relation between number of carbon atoms and band gap is verified.It is of interest to see how the properties of COFs change depending on their composition of different secondary building units, that is, for different connectors and linkers. PDOS of COFs built from type-In summary, we have designed 2D COFs of various topologies, by connecting building blocks of different connectivity and performed DFTB calculations to understand their structural, energetic and electronic properties. We have studied each COF in high-symmetry AA and AB as well as low-symmetry inclined and serrated stacking forms. The optimized COF structures exhibit different interlayer separations and band gaps in different kinds of layer stackings; however, the density of states of a single layer is not significantly different from that of a multilayer. The alternate shifted layers in AB, serrated and inclined stackings cause less repulsive orbital interactions within the layers, which result in shorter interlayer separation compared to AA stacking. All the studied COFs show semiconductor-like band gaps. We also have observed that larger number of carbon atoms in the unit cells in COFs causes smaller band gaps and vice versa. Energetic studies reveal that the studied structures are stable; however, notable difference in the layer stacking is observed from experimental observations. This result is also supported by simulated XRDs.We have optimized the atomic positions and the lattice parameters for all studied COFs. All calculations were carried out at the Density Functional Tight-Binding (DFTB) –21 level−4 eV/Å was applied. The optimization, using Γ-point approximation, was performed with the deMonNano code on 2×2×4 supercells. Some of the monolayers were also optimized using the DFTB+ code on elementary unit cells in order to validate the calculations within the Γ-point approximation. The number of k-points has been determined by reaching convergence for the total energy as a function of k-points according to the scheme proposed by Monkhorst and Pack [Periodic boundary conditions were used to represent frameworks of the crystalline solid state. The conjugate–gradient scheme was chosen for the geometry optimization. The atomic force tolerance of 3 × 10and Pack . Band stand Pack –29.We have also performed first-principles DFT calculations at the PBE  /DZP 3131 level Supporting Information supplies detailed data of calculated COF parameters and crystal information files (CIFs) of some of the studied COF structures.File 1Detailed data of calculated COF parameters.File 2CIFs for selected structures.


Arabidopsis thaliana mutants of topoisomerase 3α and RMI1 are also sterile due to extensive chromosome breakage in meiosis I, indicating that both proteins are essential for meiotic recombination in plants. AtRMI1 harbours an N-terminal DUF1767 domain and two oligosaccharide binding (OB)-fold domains. To define specific roles for these individual domains, we performed complementation experiments on Atrmi1 mutants with an AtRMI1 full-length open reading frame (ORF) or deletion constructs lacking specific domains. We show that the DUF1767 domain and the OB-fold domain 1 are both essential for the function of AtRMI1 in DNA cross-link repair as well as meiotic recombination, but partially dispensable for somatic HR suppression. The OB-fold domain 2 is not necessary for either somatic or meiotic HR, but it seems to have a minor function in DNA cross-link repair.Recombination intermediates, such as double Holliday junctions, can be resolved by nucleases or dissolved by the combined action of a DNA helicase and a topoisomerase. In eukaryotes, dissolution is mediated by the RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RecQ-mediated genome instability 1 (RMI1). Throughout eukaryotes, the RTR complex is involved in DNA repair and in the suppression of homologous recombination (HR) in somatic cells. Surprisingly,  The resolution of recombination intermediates, such as double Holliday junctions (dHJ), by endonucleases is an indispensable step for the proper segregation of homologous chromosomes in meiosis and to ensure genomic stability in somatic cells. The dissolution mechanism by the RTR complex is an alternative way to process recombination intermediates, such as dHJs ,2. This BLM, WRN and RECQ4 genes are the cause of severe hereditary diseases, namely, Bloom  as well as type IA topoisomerases  were identified as proteins involved in this pathway ,13,14.Interestingly, a third protein named RecQ-mediated genome instability 1 (RMI1)  was found to be required for the dissolution mechanism. These three proteins form the evolutionarily highly conserved RTR complex ,15–17.in vitro , the RTR complex is composed of the RecQ helicase RECQ4A, the type IA topoisomerase TOP3A and RMI1 , a first oligonucleotide/oligosaccharide binding-fold (OB-fold) domain (OB1) followed by a second OB-fold domain (OB2) in the C-terminal part of the protein. The overall sequence identity between the RMI1 homologue in Arabidopsis and humans is low, but the domains are highly conserved. The function of the DUF1767 domain is still unclear, but it is thought to be important for the proper folding of the protein  and Atrmi1-2 (SALK_094387) of A. thaliana ecotype Col-0 were used. These mutant lines from the Salk collection . The second PCR was performed with one primer upstream of the deletion and one primer located within the deletion (5′-ATGTGTGATTTTGGCTGAAC-3′/5′-CTAAACGAGTACATTGTCAG-3′). To detect the wild-type allele of the Atrmi1-2 line, one pair of primers was located upstream and downstream of the insertion site (5′-TTCACCATAGCCGAGTTAC-3′/5′-AGAAGCTCATACGTAGACTG-3′). The second primer pair contained one primer binding to the AtRMI1 locus and one T-DNA-specific primer (5′-TTCACCATAGCCGAGTTAC-3′/5′-TCGGAACCACCATCAAACAG-3′).Two primer pairs were used to genotype each of the AtRMI1 or the AtRMI1 deletion variants are based on the cDNA sequence of AtRMI1 . In theAtrmi1-1, Atrmi1-2 and wild-type plants  was performed via the floral dip method (Agrobacterium-mediated transformation of p method . After tActin2 gene (At3g18780) (5′-CAGATGCCCAGAAGTCTTG-3′/5′-GTGCTGTGATTTCTTTGCTC-3′) as an internal standard (Atrmi1-2 mutant or an amplicon of 200 bp in the Atrmi1-1 mutant (5′-ATTCACCGAGCAGCATCCAC-3′/5′-TACACCGCCTGAATCTGAAC-3′). The gene expression calculations were performed with the LightCycler 480 SW 1.5 software (Roche Diagnostics GmbH). The relative quantification was done after the PCR efficiency calculation from standard curves of both the target and reference amplicons that were generated from serial dilutions of wild-type cDNA. To determine Cp calling, the second derivative maximum method was used.Total RNA was extracted from 2-week-old plantlets using the RNeasy Plant Mini Kit  according to the manufacturer’s instructions. Reverse transcription was conducted using the RevertAid First-strand cDNA Synthesis Kit  according to the manufacturer’s instructions. Expression analysis was performed by quantitative PCR  with SYBR Green I Master Mix . The results were normalized using the constitutively expressed standard . The priSensitivity assays were performed as previously described . After 12HPO4 (pH 7); 1 ml of 5% sodium azide, 2.5 ml of 1% X-GlcA, 100 mg of 5-bromo-4-chloro-3-indolyl-b-d-glucuronide dissolved in 10 ml of DMF] for 2 days at 37°C and an extraction of plant pigments in 70% EtOH at 60°C overnight, which facilitates the quantification of blue sectors using a binocular microscope.The HR assays using the IC9 reporter construct were performed as described ,33 by trThe chromatin staining of the pollen mother cells was performed as described ,38. PrimPreviously published data for the human RMI1 homologue revealed the existence of three conserved domains ,22,30. AA. thaliana RMI1 gene (At5g63540) contains an ORF of 2247 bp (AY735746) that encodes a protein of 644 aa , RMI1ΔDUF (550 aa), RMI1ΔOB1 (619 aa), RMI1ΔOB2 (484 aa) and RMI1ΔDUFΔOB1 (525 aa) . Therefore, a lack of complementation of the mutant phenotype cannot be due to insufficient gene expression. A direct comparison of the protein concentrations could not be performed, owing to the lack of an AtRMI1-specific antibody.All constructs were cloned between the natural (525 aa) . To ensuAtrmi1 mutants that we characterized previously  or the alkylating agent MMS (60 ppm) in comparison with untreated plants. After 1 week of growing on solid medium, the plants were transferred to liquid medium and treated with cisplatin or MMS for two additional weeks. The complementation of the enhanced cisplatin and MMS sensitivity of the Atrmi1-2 mutant was achieved by the expression of the wild-type AtRMI1 construct  and MMS sensitivity  compared with the mutant line. The recombinant proteins RMI1ΔDUF and RMI1ΔOB1 could neither complement the enhanced sensitivity against cisplatin nor the enhanced sensitivity against MMS. For both constructs, the four tested lines are similar in their sensitivity to the Atrmi1-2 mutant line . The plantlets were transferred into liquid medium after 1 week of growth on solid medium. After another week with or without cisplatin treatment in liquid medium, the plants were fixed and stained in a sodium azide and X-GlcA solution. In both the spontaneous and cisplatin-induced HR assays, the mutant phenotype could be successfully complemented by the expression of the wild-type RMI1 construct and the RMI1ΔOB2 construct . On the other hand, the deletion of the DUF1767 domain and the OB-fold domain 1 in the N-terminal part of AtRMI1  both resulted in an intermediate level of HR events compared with the HR frequency of the Atrmi1-2 mutant and the wild-type . After treatment with cisplatin, the HR frequency of all four tested lines expressing RMI1ΔOB1 is significantly different from wild-type  as well as from rmi1-2 . In the lines expressing RMI1ΔDUF, only line #5 shows no significant difference from wild-type, whereas lines #6, 7 and 8 have a significantly higher HR frequency compared with wild-type . Furthermore, the HR frequencies of lines 5 and 7 are also significantly lower than the mutant’s . It is noteworthy that, as in the case of cisplatin and MMS hypersensitivity, no partial complementation of the Atrmi1-2 mutant phenotype could be obtained by transformation with the RMI1ΔDUF and RMI1ΔOB1 constructs (see earlier in the text).The enhanced HR frequency of the line IC9 . This cos formed E. Each rs formed ; for a rs formed ]. The coisplatin  and in uAtrmi1-2 mutant, four different independent transgenic lines were isolated and analysed. The expression of the recombinant protein RMI1ΔDUFΔOB1 did not significantly change the frequency of HR of the Atrmi1-2 mutant either with or without cisplatin treatment  or with cisplatin treatment . The fact that Atrmi1 plants expressing an AtRMI1 protein without both the DUF1767 and OB domain 1 show a recombination frequency that is higher than that of plants expressing an AtRMI1 where only one of the domains is missing  indicates that both domains have at least partially non-overlapping functions in the suppression of somatic HR.The previous results indicated that both N-terminal domains DUF1767 and OB1 have some function in the suppression of somatic HR. However, both could be involved in the same type of reaction that would require AtRMI1 for some but not all forms of HR suppression detected by our assay system. On the other hand, both domains might contribute, at least partially, in an independent way to the suppression phenotype so that the loss of both domains would lead to a complete loss of suppression. To discriminate between these possibilities, we generated a construct missing both the DUF1767 domain and the OB-fold domain 1 . After treatment . On the A. thaliana RMI1 is also accompanied by a meiotic recombination defect leading to sterile plants. Therefore, the lines expressing the recombinant proteins RMI1, RMI1ΔDUF, RMI1ΔOB1, RMI1ΔOB2 and RMI1ΔDUFΔOB1 were tested for their ability to complement the sterile phenotype and the meiotic arrest of the Atrmi1-1 mutant line. The expression of the different constructs was measured by quantitative RT-PCR . To define the role of the individual domains in meiotic recombination, we analysed the number of fertile Atrmi1-1 T1 plants after the transformation of hemizygous Atrmi1-1 plants with the AtRMI1 full-length construct and the four deletion constructs . Transformed plants were selected by PPT resistance mediated by the inserted T-DNA, and the AtRMI1 mutation was genotyped by PCR. The fertility of homozygous Atrmi1-1 plants was checked by counting the number of seed-bearing T1 plants in the greenhouse . The remarkable meiotic phenotype of the Atrmi1-1 mutant becomes apparent in anaphase I of the first meiotic division. In comparison with anaphase I in the pollen mother cells of wild-type plants   ,44. The in vitro .Atrmi1 mutant plants. As even small changes in amino acid sequences can influence folding and hence structure and activity of proteins, one has to be careful in the interpretation of data acquired with such constructs. We were not able to directly measure the expressed proteins in planta, but we are confident that the kind of intermediate effects on HR frequency seen in our experiments are not likely due to a general destabilization of the proteins involved. Furthermore, similar experiments have been performed with RMI1 and other proteins of the RTR complex in vitro and in vivo, showing stable expression of protein variants (Atrmi1 mutant lines used in this study have to be considered. Line Atrmi1-2, which was used for somatic analyses, is able to form a transcript of the gene 5′ of the inserted T-DNA (Atrmi1-2, can complement some phenotypes of this mutant. Together with the observation that the full knockout line Atrmi1-1 displays a comparable somatic phenotype to Atrmi1-2, it is highly likely that there is only some minor activity left in Atrmi1-2. Furthermore, the fact that we were able to complement the Atrmi1-2 DNA repair and HR phenotypes to wild-type levels can be taken as a strong indication that the mutant phenotype is not due to the expression of a negatively complementing protein fragment.One major approach of this work was to express AtRMI1 protein variants with deleted domains in variants ,30,45. Aed T-DNA . This frin vivo domain analysis indicate that AtRMI1 is involved, at least partially, in a number of different steps of DNA cross-link repair, suppression of somatic HR and meiotic recombination. The DUF1767 domain and the OB-fold domain 1 are both essential for DNA cross-link repair. Moreover, the OB-fold domain 2 also plays some role in this pathway. Owing to random DNA integration following transformation of A. thaliana, rarely all transformed lines are phenotypically similar to each other. In the case of the lines expressing construct RMI1ΔOB2, two of the four lines tested were not different from the mutant, whereas the other two were able to render the plants more resistant to cisplatin and MMS treatment. As some transgenic lines are expected to be located in transcriptionally silent loci, we tested the expression of the RMI1 construct in all four lines. However, expression levels did not correlate with cisplatin or MMS sensitivity levels. Therefore, differing transcriptional activities cannot be the reason behind the differences. As specific insertion loci were not determined for each line, there might be unknown kinds of locus-specific indirect effects. Nevertheless, at least half of the tested lines were able to repair cisplatin or MMS-induced DNA damage almost as efficiently as wild-type.The results of our Thus, all three domains are involved in this type of repair reaction. This contrasts with the behaviour in somatic and meiotic recombination. The OB-fold domain 2 is dispensable for both types of recombination reactions. Nevertheless, our results indicate that the role of AtRMI1 in both pathways is not identical. Although individually the DUF1767 as well as OB-fold domain 1 are absolutely essential for the progression of meiosis, a partial suppression of somatic recombination can occur if one or the other domain is not functional. This shows that the AtRMI1 DUF1767 domain is not simply an N-terminal extension of the first OB-fold domain, but that it must possess a function distinct from the OB1 domain.Atrmi1 mutants with a recombinant protein that does not contain the OB-fold domain 2. According to these results, the interaction of AtRMI1 with a putative AtRMI2 is dispensable for recombination functions. HsRMI2 could be identified as a fourth essential component of the RTR complex in humans but not in yeast (A. thaliana, which possesses a similar predicted structural organization needed for the interaction of HsRMI2 and HsRMI1 (in planta and is involved in certain pathways of DNA repair. Therefore, it will be an interesting task to define the biological role of AtRMI2 in detail.AtRMI1 is involved in different types of reactions in DNA repair and HR. During these reactions, it interacts to different extents with other proteins via its respective domains. Our analysis shows that the OB-fold domain 2 from AtRMI1 is dispensable for HR in somatic and meiotic cells. We found that it is possible to fully complement both the elevated frequency of HR in somatic cells and the meiotic defect of the in yeast ,22. An Rd HsRMI1 ,31. As tThe fact that we detected partial complementation in some assays can be taken as a hint that there might be more than just one pathway addressed with this assay. This applies for the role of OB-fold domain 2 in DNA repair as well as the DUF1767 and the OB-fold domain 1 in the suppression of somatic HR. All this indicates that RMI1 has a much more complex role in mediating DNA processing enzymes than originally anticipated. In addition to dissolving dHJs, the RTR complex is involved in other steps of HR, such as the resection of double-stranded DNA ends in yeast. Interestingly, no active topoisomerase is required for this function . It has Supplementary Data are available at NAR Online.German Research Foundation DFG [Pu 137/11] and the European Research Council ERC [ERC-2010-AdG_20100317 COMREC]. Funding for open access charge: German Research Foundation [Pu 137/11].Conflict of interest statement. None declared.


In the past decade much has been learned about how Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) folds and misfolds as the etiologic cause of cystic fibrosis (CF). CFTR folding is complex and hierarchical, takes place in multiple cellular compartments and physical environments, and involves several large networks of folding machineries. Insertion of transmembrane (TM) segments into the endoplasmic reticulum (ER) membrane and tertiary folding of cytosolic domains begin cotranslationally as the nascent polypeptide emerges from the ribosome, whereas posttranslational folding establishes critical domain–domain contacts needed to form a physiologically stable structure. Within the membrane, N- and C-terminal TM helices are sorted into bundles that project from the cytosol to form docking sites for nucleotide binding domains, NBD1 and NBD2, which in turn form a sandwich dimer for ATP binding. While tertiary folding is required for domain assembly, proper domain assembly also reciprocally affects folding of individual domains analogous to a jig-saw puzzle wherein the structure of each interlocking piece influences its neighbors. Superimposed on this process is an elaborate proteostatic network of cellular chaperones and folding machineries that facilitate the timing and coordination of specific folding steps in and across the ER membrane. While the details of this process require further refinement, we finally have a useful framework to understand key folding defect(s) caused by ΔF508 that provides a molecular target(s) for the next generation of CFTR small molecule correctors aimed at the specific defect present in the majority of CF patients. Cystic fibrosis (CF) is one of a growing number of human diseases caused by inherited mutations that disrupt protein folding. It is caused by dysfunction of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a cAMP-regulated ion channel that resides in the apical membrane of epithelial cells Riordan, . CFTR dyCystic fibrosis transmembrane conductance regulator is a 1480 amino acid polytopic glycoprotein in the ABC transporter family (ABCC7) that contains two six-spanning transmembrane (TM) domains (TMD1 and TMD2) that form the channel pore, two cytosolic nucleotide binding domains (NBD1 and NBD2) that drive channel gating, and an intrinsically unstructured regulatory (R) domain that controls channel activity via PKA-mediated phosphorylation Figure , suggestThis review will focus on the current state of knowledge as to how CFTR domains fold, how they interact, how mutations alter this process, and how misfolded conformations are distinguished from native structure by cellular chaperone machinery.M. jannaschii (SecYEβ) have suggested that TMs exit the translocon via a lateral cleft between Sec61α TMs2-3 and TMs7-8 along one side of the PCC  that sequentially open the translocon pore into the ER lumen to initiate translocation and close the pore to terminate translocation and direct peptide segments into the cytosol. Such a mechanism would maintain ER integrity while essentially stitching TM segments into the bilayer via coordinated structural changes at the lumenal and the cytosolic faces of the RTC Johnson, .Homology models predict that CFTR exhibits a complex domain swap structure in which two six-spanning helical bundles containing TMs1-2, 9-12 and TMs7-8, 3-6 are twisted around a central ion-conducting pore  and strong type I SA (TM2) activities. Subsequently, TM3-4 and TM5-6 insert into Sec61 as helical hairpins to translocate short ECL2 and ECL3 loops. An interesting and currently unresolved question is whether TMs or TM pairs partition freely into the bilayer from the small Sec61αβγ pore, if they remain associated with Sec61 and/or other translocon proteins during subsequent helical packing. This question is particularly relevant in light of the mature domain swap structure where TM1-2 ultimately bundles with TM9-12 and TM3-6 with TM7-8. Given the prevalence of ionizable residues in TMs1, 2, 3, and 6, final assembly likely requires precise alignment of TMs prior to complete integration into the lipid bilayer.When TM6 terminates translocation NBD1 cotranslationally passes beneath the base of the ribosome into the cytosol. Several features of CFTR suggest that during this process, the ribosome transiently disengages from Sec61 to allow folding of the cytosolic domains. CFTR TMD1 helices are predicted to extend ∼40–50 Å from the membrane, and these extensions appear to provide a preliminary docking site for NBD1 prior to synthesis of TMD2  and stop transfer  sequences. As TM7 emerges from the ribosome, it efficiently directs membrane targeting and ECL4 translocation. TM8, which is separated from TM7 by ∼31 residues, terminates translocation, and redirects ICL3 in the cytosol as expected. Interestingly, TM8 functions as an efficient stop transfer only when it is normally paired with TM7, but not in a heterologous context , coincident insertion of helical hairpins , cooperativity for topogenic determinant function (TM7-8), and regulated integration into the ER membrane. Reasons underlying these distinct translocation mechanisms are only beginning to be understood, but evidence suggests that different folding pathways have functional implications. For example, replacement of ionizable residues in TM1 (E92A and K95A) converts TM1 to a strong signal anchor sequence, thus favoring cotranslational topogenesis, but disrupts CFTR function  between Donor and Acceptor probes that are cotranslationally incorporated at distant sites in primary sequence but which become proximal to one another as the protein folds. Because FRET efficiency is highly sensitive to changes in distance on a scale of ∼10–80 Å, changes in FRET that occur at increasing chain lengths provide a sensitive readout for nascent chain compaction and folding. Using this approach, Khushoo et al.  showed tBased on the time required for CFTR to exit the ER, CFTR domain assembly takes ∼30–120 min. This suggests a hierarchical process in which domain folding begins cotranslationally and is followed by posttranslational formation of domain–domain contacts  at various stages of folding Skach, . Major cin vitro possibly by reducing off-pathway folding events  caused by ΔF508 in the majority of CF patients. Within NBD1 itself, removal of Phe508 decreases folding efficiency and renders the domain susceptible to unfolding, denaturation, and aggregation at physiologic temperatures, possibly as a direct result of destabilizing the α-helical subdomain. Absence of Phe508 also disrupts the interaction between NBD1 and ICL4 (within TMD2), which distorts TMD structure and interferes with channel gating. Defects in NBD1 and the NBD1–ICL4 interface are both recognized by quality control machinery, and correction of both is necessary and sufficient to restore trafficking and function to near WT levels. Importantly, partial correction of ΔF508 CFTR folding can be achieved by a variety of means: cis-acting suppressor mutations, manipulation of the proteostatic network, or small molecule correctors. Moreover, combinations of these maneuvers are now able to achieve near WT levels of surface expression and function. Thus, it is increasingly attractive to target the next generation of CFTR small molecule correctors to specific defects that will optimize synergy in correction mechanisms. While the most precise targets reside within the CFTR molecule itself, i.e., NBD1 and the NBD1–TMD2 interface, it is also possible that other clinically beneficial targets will be developed in the years to come, which will undoubtedly be driven by increasing resolution of the folding problem.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The actin cytoskeleton is essential for many physiological processes of eukaryotic cells. The emergence of new actin fibers is initiated by actin nucleators. Whereas most of them are evolutionary old, the cordon-bleu actin nucleator is classified as vertebrate specific.Nematostella vectensis.Using sensitive methods for sequence similarity detection, we identified homologs of cordon-bleu not only in non-vertebrate chordates but also in arthropods, molluscs, annelids and platyhelminthes. These genes contain only a single WH2 domain and therefore resemble more the vertebrate cordon-bleu related 1 protein than the three WH2 domain containing cordon-bleu. Furthermore, we identified a homolog of the N-terminal, ubiquitin like, cobl domain of cordon-bleu in the cnidarian Drosophila melanogaster could facilitate its experimental characterization.Our results suggest that the ur-form of the cordon-bleu protein family evolved already with the emergence of the bilateria by the combination of existing cobl and WH2 domains. Following a vertebrate specific gene-duplication, one copy gained two additional WH2 domains leading to the actin nucleating cordon-bleu. The function of the ur-form of the cordon-bleu protein family is so far unknown. The identification of a homolog in the model organism  The actin cytoskeleton is one of the hallmarks of eukaryotic cells. It is involved in processes like movement, phagocytosis and morphogenesis, to name just a few . AlthougBranchiostoma floridae . In the first iteration, hits were found in Saccoglossus kowalevskii  and the sea urchin Strongylocentrotus purpuratus . Surprisingly, also proteins of arthropods, e.g. the honey bee Apis mellifera , and of Lophotrochozoa, namely the oyster Crassostrea gigas , the sea hare Aplysia californica  and the annelid Capitella teleta , were hit. The species most distantly related to vertebrates identified in this iteration was Capsaspora owczarzaki, belonging to the Ichtyosporea . In the next iteration, the profile showed significant similarity to further arthropod proteins including Drosophila melanogaster "proximal to raf" . Finally, first bona fide ubiquitin like proteins  were hit. Back searches with e.g. the Capitella teleta sequence identified additionally a protein in the platyhelminth Clonorchis sinensis . We thus conclude that the cobl domain evolved at least with the emergence of the bilateria.WH2 domains are short and divergent and thus hard to identify with standard sequence analysis approaches. We therefore in the first step focused on a second conserved domain in the N-terminus of cordon-bleu, the cobl domain. This domain belongs to the ubiquitin fold and its structure has been solved (PDB 2DAJ). A PSI-Blast search  with thiTrichoplax adhaerens[Amphimedon queenslandica[Hydra magnipapillata[Nematostella vectensis[Mnemiopsis leidyi[Nematostella vectensis . Still, this single occurrence leaves the presence of a cobl domain in the non-metazoan eukaryote Capsaspora owczarzaki enigmatic.To identify possible non-bilaterian members of the domain, we performed Hidden Markov Model searches  with an adhaerens), a sponnslandica), cnidarpapillata and Nemavectensis) and Cteis leidyi). Indeed-6). Therefore, these arthropod sequences contain not only the cobl, but also an additional C-terminal WH2 domain. With the same approach, we were able to identify a single WH2 domain at the C-terminus of the oyster protein (E=0.00025). Next, we aligned a HMM based on vertebrate cordon-bleu proteins against one based on the putative Drosophila homologs. In addition to the cobl domain, a significantly (E=0.065) similar region was identified between the C-terminus of both alignments. Taking the human cordon-bleu as reference, this covered positions 1234 to 1247 and thereby at least parts of the C-terminal WH2 domain. This indicates a single WH2 domain in the C-terminus of the Drosophila "proximal to raf" proteins. Despite the sensitivity of these approaches, no WH2 domain was predicted in the Nematostella vectensis protein containing a cobl domain. Contrasting, InterPro identified a WH1  domain in position 75–186. The cobl domain containing Capsaspora owczarzaki protein also lacks a WH2 domain, but contains a WW, a PTB and a CRIB domain. Most interestingly, the latter is also found in WASp proteins.But, do these proteins indeed belong to the cordon-bleu protein family? In vertebrates, a paralog of cordon-bleu, cordon-bleu related 1, exists. Whereas the first contains three C-terminal WH2 domains, the latter contains only one. We therefore set out to identify WH2 domains in the cobl domain containing proteins using sensitive HMM to HMM alignments ,21. IndeTaken together, we have shown that the evolutionary roots of the cordon-bleu (related) protein family lie before the first emergence of vertebrates. Its absence in some major metazoan lineages like the Nematoda can be explained by lineage specific losses. Thus, our results suggest the following evolutionary history of cordon-bleu proteins. First, the N-terminal cobl domain evolved from an existing ubiquitin fold. With the emergence of the bilateria, this domain was combined with a single WH2 domain. Finally, with the emergence of the vertebrates, a gene duplication evolved the cordon-bleu related 1 and the cordon-bleu genes. In the latter, two additional WH2 domains were acquired. We were not able to predict whether these new domains arose via an internal duplication or were acquired from another protein as the sequences were too short to calculate a reliable phylogeny.Drosophila melanogaster could enable the functional characterization of the ur-form of the cordon-bleu protein family. Following, the adaptations leading to the vertebrate specific function of cordon-bleu could be traced. Thus, the cordon-bleu protein family could become a test case to study functional changes following gene duplications.To perform as actin nucleator, a protein has to bring different monomeric actin molecules in close proximity. In cordon-bleu, this is achieved by the binding of actin to each of the three WH2 domains. Contrasting, cordon-bleu related 1 as well as the ur-form pre-dating the duplication contain only a single WH2 domain. Therefore, it is unlikely that these molecules have actin nucleation capabilities. Thus, this function evolved following the gene duplication. The identification of a candidate ortholog in the model organism The authors declared that they have no competing interests.JS designed the study, performed analyses and wrote the Manuscript. NT performed analyses. Both authors read and approved the final manuscript.


PEG3 has been identified as partner of the E3 ubiquitin-protein ligase Siah1, an association we sought to investigate. An efficient bacterial recombinant expression system of the human PEG3-SCAN domain was prepared and crystals appeared spontaneously when the protein was being concentrated after purification. The structure was determined at 1.95 Å resolution and reveals a polypeptide fold of five helices in an extended configuration. An extensive dimerization interface, using almost a quarter of the solvent accessible surface, and key salt bridge interactions explain the stability of the dimer. Comparison with other SCAN domains reveals a high degree of conservation involving residues that contribute to the dimer interface. The PEG3-SCAN domain appears to constitute an assembly block, enabling PEG3 homo- or heterodimerization to control gene expression in a combinatorial fashion.Human paternally expressed gene 3 protein (PEG3) is a large multi-domain entity with diverse biological functions, including acting as a transcription factor. PEG3 contains twelve Cys When a knockout model was assessed, growth retardation, increased levels of body fat, impairment of maternal behaviour and changes in male sexual behaviour were noted Human paternally expressed gene 3 protein (PEG3) is a large multi-functional protein of nearly 1600 amino acids with a mass of about 165 kDa. The protein carries twelve zinc finger DNA-binding motifs, which are an important component of transcription 2-His2 Krüppel-type motifs frequently contain other domains, which are implicated in subcellular localization, protein-protein and protein-DNA interactions. These include the Krüppel-associated box domain, the poxvirus and zinc finger (POZ), alternatively known as the BTB , and the SCAN domain, sometimes called the leucine rich motif SRE-ZBP, Ctfin51, AW-1 (ZNF174), and Number 18) 2-His2 Krüppel-type motifs allows them to interact in a combinatorial fashion to control gene expression Proteins that carry Cysα-helicesPreviously, two NMR and two crystal structures of SCAN domains have revealed the structural topology, which is constructed around five Here, we report the structure of the isolated SCAN homodimer from the human PEG3 protein (PEG3-SCAN). Comparisons with other SCAN structures in the Protein Data Bank (PDB) and the importance and contributions of residues involved in PEG3-SCAN dimerization are discussed, and provide insight into homo- and heterodimer assembly.Escherichia coli Bl21 (DE3) Gold cells (Novagen) for expression. Cells were grown in Luria-Bertani media at 37°C to an OD600 of ∼0.6, followed by induction with 0.2 mM IPTG. Cells were then grown overnight at 22°C and harvested by centrifugation at 3,500 g for 30 minutes at 4°C. The cell pellet was suspended in lysis buffer  containing DNase I (0.1 mg), with a single protease inhibitor tablet (Roche) and lysed using the French Press . The extract was centrifuged at 37,500 g for 30 minutes at 4°C to pellet insoluble debris. The supernatant was loaded onto a 5 mL HisTrap HP column  and a linear imidazole concentration gradient from 20 mM to 1 M was applied. The PEG3-SCAN sample eluted from the column at approximately 200 mM imidazole. Fractions containing the recombinant protein were pooled and dialyzed in buffer , prior to addition of TEV protease to remove the His-tag. TEV protease was prepared in-house by Keri Barrack (University of Dundee). 1 mg of TEV was used per 20 mg of protein in a cleavage reaction, which was performed at room temperature for three hours. After TEV digestion, three non-native residues (Gly-His-Met) remained at the N-terminus of the product. The protein was passed through a second nickel-affinity column, which removed uncleaved protein and the His-tagged TEV protease. Gel filtration  was used as a final purification step. The column had previously been calibrated with molecular weight standards, blue dextran , thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29.5 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa). PEG3-SCAN eluted from the size exclusion column as a single symmetric peak with a mass of approximately 28 kDa. The theoretical mass of PEG3-SCAN is 11,434 Da; therefore PEG3-SCAN forms a homodimer in solution. The purity of the sample was checked further by SDS-PAGE and mass spectrometry . The single protonated species as observed by mass spectrometry was 11,432 Da, in close agreement with the theoretical mass. Protein concentration was determined spectrophotometrically using a theoretical molar extinction coefficient of 16,960 M−1 cm−1The gene encoding the SCAN domain of human PEG3  was purchased in the pUC57 vector (GenScript). The gene was subsequently sub-cloned into a modified pET15b vector (Novagen), designed to express the protein of interest with an N-terminal hexa-His tag and a Tobacco Etch Virus (TEV) protease cleavage site. The sequence-verified clone  was transformed into 15N-labeled Siah1 for NMR studies were prepared and purified using a similar protocol to that for PEG3-SCAN, except that isotopically enriched Siah1 was expressed in the minimal media. A single sharp peak was observed for Siah1 on GF column with a mass of around 39 kDa. This value matches closely to the weight of Siah1 homodimer, as the theoretical mass of a monomer is 21,897 Da. The presence of the Siah1 homodimer was confirmed by size exclusion chromatography (SEC) coupled with multi-angle light scattering. This supports previous studies showing Siah1 is a dimeric protein −1 cm−1oC, before it was run on a GF column . The NMR experiment was done under the following conditions, where 100 µM of 15N-labeled Siah1 was mixed with 100 µM of unlabeled PEG3 in 50 mM HEPES, pH 7.5, 50 mM NaCl, and 5% D2O buffer. The chemical shift perturbations in the 1H-15N HSQC of Siah1 were monitored upon addition of PEG3.The gene coding for part of human Siah1 without the RING domain  was purchased in the pUC57 vector (GenScript). The gene fragment was transferred into the pET15b vector (Novagen) for recombinant expression. Siah1 and oC in the buffers tested again with a profile indicative of a folded protein.Differential scanning fluorimetry (DSF) was used to investigate the influence of different buffers on the thermal stability of the samples. DSF suggested the presence of a globular SCAN domain, which displayed a melting temperature of 52°C in a number of buffers. Since no buffer appeared to enhance stability the protein was left in the GF buffer . The melting temperature of Siah1 was 64−1 were mixed in sitting-well plates with 100 nL of reservoir solution against 70 µL of the same reservoir solution. Crystals appeared within a day in numerous conditions that contained polyethylene glycol (PEG) of different molecular weight. These crystallization conditions were scaled up from these nano-drops to micro-drops of total volume 4 µL. However, the rod-shaped crystals that were observed were small, approximately 0.1×0.02×0.02 mm, and gave poor diffraction. While preparing more protein for use in crystal optimization it was observed that larger crystals actually formed spontaneously when the protein was concentrated in the GF buffer using Vivaspin 20 concentrators with a 3,000 MW cut off (Sartorius Stedim Biotech). Hexagonal bipyramid crystals, reaching 0.2×0.2×0.2 mm dimensions, formed within minutes  and several commercially available screens (Hampton Research). 100 nL of protein sample at ∼16.5 mg mL minutes . The aveiMOSFLMAIMLESS from the CCP4 program suite PHASERREFMAC5COOTB-factor restraint weights. Neither non-crystallographic symmetry (NCS) restraints nor TLS (Translation/Libration/Screw) were used in refinement. A number of ligands  were included in the model on the basis of the difference density and chemical environment, and refined successfully. These molecules are likely to be decomposition products or impurities of the PEG200 cryoprotectant. The final model also includes multiple side chain conformers and water molecules. The stereochemistry of the model was checked using MOLPROBITYPISA  radiation and an AFC11 Saturn 944+ CCD detector (Rigaku). The data were indexed and integrated with P65, with a VM value of 2.44 Å3 Da−1 and solvent content of approximately 50% for an asymmetric unit comprising two polypeptide chains. The polypeptides are arranged as a symmetrical dimer consistent with the GF results obtained during protein purification and also with previously solved structures of SCAN domains. The crystals diffract to a resolution of 1.95 Å and the majority of the residues are located within well-defined electron density, apart from a few residues at the C-terminus. In addition, the final model contains two extra residues (His and Met) at the N-terminus, which are remnants from proteolytic cleavage of the histidine tag. A Ramachandran plot indicates that 98.4% of the amino acids are located in the most favoured region with no outliers.Crystals of human PEG3-SCAN belong to space group LSQKABThe human PEG3-SCAN domain folds as an extended V-shaped structure, with approximate overall dimensions of 50×25×25 Å. Each arm of the V-shape is approximately 35 Å in length. The subunit comprises five α helices and the assignment of secondary structure onto the sequence is presented in PISA2 of which 1700 Å2 are buried within the dimer. Therefore, about 23% of the surface area of each monomer is involved in dimerization. The free energy of dissociation (ΔGdiss) is estimated as 19.4 kcal mol−1, and suggests that this assembly is thermodynamically stable, consistent with the observation of a stable dimer in solution. Similar values are observed for other SCAN structures. For example, the interface area and ΔGdiss for the Znf24 dimer (PDB code 3LHR) are 23% and 21.8 kcal mol−1, respectively.The total surface area of a subunit, calculated with At present there are four SCAN domain structures in the PDB, two crystal structures and two determined by solution NMR. Sequence conservation of these four with human PEG3-SCAN is presented in The human PEG3-SCAN homodimer is held together by an extensive network of hydrogen-bonding, salt-bridge interactions and van der Waals forces. Even though the overall sequence identity among the five known SCAN structures is only 40–50% , the keySome of the interactions noted in the PEG3-SCAN dimer are absent from the other structures. For example, a hydrogen bond donated from the side chain of Tyr94 on one subunit to the carbonyl of Pro60  on the pWhilst most of the dimer interface excludes water molecules, with the notable exception described above, at the periphery of the SCAN dimer there are five that are involved in mediating subunit-subunit interactions (data not shown). This does not appear to be a major factor in stabilizing the dimer given the high percentage of the surface area involved in direct association as described above.The dimer interface includes important stabilizing contributions from hydrophobic residues. A hydrophobic patch comprising Phe49, Trp74, Leu75, Ile85, Ile86 and Leu89, from α2 and α3, make van der Waals interactions to equivalent residues in the partner subunit . In anotThe strictly conserved pair of residues Pro60 and Pro99, already mentioned as contributing to the inter-subunit hydrophobic associations, are positioned at the start of α2 and α4 respectively , and lik2-His2 zinc finger containing transcription factors that contain a protein-protein binding BTB/POZ domain. Self-oligomerization of this domain in the GAGA transcription factor promotes DNA binding affinity and specificity 2-His2 zinc finger proteins. To date, there are no data to suggest that SCAN domains participate directly in transcriptional activation or repression. The possibility that PEG3 could homodimerize through the SCAN domain suggests however that PEG3 binds to at least some target genes as a homodimer. In particular, this might be a prerequisite for PEG3 binding palindromic DNA motifs. Consistent with this is the finding that the Zfp206 transcription factor can form a homodimer using its SCAN domain and binds to a palindromic sequence Transcription factors form complexes that regulate the expression of target genes. For example, the proto-oncogene Jun, belonging to the basic region-leucine zipper (bZIP) family, regulates gene transcription in a specific manner by forming homo- and heterodimers. It is the combination of dimerization state and binding partners that determine activity and DNA-binding site preferences 2-His2 zinc finger motifs, while in two other isoforms the SCAN domain is absent. Thus, the presence or absence of the SCAN domain and varying number of zinc finger motifs provides a potential additional control mechanism. A number of SCAN domain members have been reported to selectively interact with other SCAN members The DNA binding selectivity and affinity of PEG3 might depend on its specific isoform. To date, there are four known PEG3 isoforms. One isoform contains only two CysIt has been suggested that some SCAN domains act as specific cellular localization sequences. For example, the SCAN domain of Zfp42 is reported to be essential for targeting the protein to promyelocytic leukemia nuclear bodies in the cell nucleus 1H–15N HSQC (heteronuclear single quantum coherence spectroscopy) spectra of 15N-labeled Siah1 upon addition of unlabeled PEG3-SCAN (data not shown) indicative of a lack of interactions between the polypeptides. The lack of an association suggests that residues outside the SCAN domain might be required for interaction with Siah1 but there is no other obvious Siah1 binding motif. The PEG3-SCAN structure reveals that the Val58-Gly59-Pro60 motif is an extended conformation immediately prior to α2. The proline is buried and involved in inter-subunit interactions, and the valine side chain tucked down towards the side of α2, away from the surface of the protein. We speculate that if this is indeed a recognition site for Siah1 then conformational changes might be required to allow for complex formation. The conditions under which we investigated the potential interaction may not have been correct to allow such changes or alternatively the presence of the Val-x-Pro sequence is coincidence. Either way, our observations suggest that the data invoking Siah1 interactions with PEG3 should be re-evaluated.It is not known whether SCAN domains interact with any other protein motifs in addition to self-association with other SCAN members. The function of PEG3 in regulation of TNF and Wnt signal transduction pathways has been implied by an ability to bind TRAF2, the E3 ubiquitin ligase Siah1 and β-catenin In summary, we have determined the structure of SCAN domain from PEG3, a predicted transcription factor, and compared it with available SCAN domain structures. Our results show this domain forms a stable homodimer and we provide an analysis of the residues forming the dimer interface. The sequence alignment and an overlay of PEG3-SCAN with available SCAN domain structures shows overall structural conservation and serves to identify key residues important to the creation of the PEG3-SCAN dimer interface.The SCAN domain of PEG3 appears to function as a convenient dimerization domain. Gel filtration chromatography and NMR studies revealed no interaction between the SCAN domain and the potential PEG3-binding protein Siah1. Future studies will be needed to determine if indeed the SCAN domain of PEG3 interacts with other SCAN family members as well as other protein motifs. The validation of binding partners would represent a crucial step towards unraveling the biological role of PEG3 itself.


C. elegans to characterize their function and mode of interaction.Proteins may evolve through the recruitment and modification of discrete domains, and in many cases, protein action can be dissected at the domain level. PDZ domains are found in many important structural and signaling complexes, and are generally thought to interact with their protein partners through a C-terminal consensus sequence. We undertook a comprehensive search for protein partners of all individual PDZ domains in C. elegans and provide a proof-of-principle for its application to the discovery of PDZ domain targets for any protein or peptide of interest.Coupling high-throughput yeast two-hybrid screens with extensive validation by co-affinity purification, we defined a domain-orientated interactome map. This integrates PDZ domain proteins in numerous cell-signaling pathways and shows that PDZ domain proteins are implicated in an unexpectedly wide range of cellular processes. Importantly, we uncovered a high frequency of non-canonical interactions, not involving the C-terminus of the protein partner, which were directly confirmed in most cases. We completed our study with the generation of a yeast array representing the entire set of PDZ domains from We provide an extensive domain-centered dataset, together with a clone resource, that will help future functional study of PDZ domains. Through this unbiased approach, we revealed frequent non-canonical interactions between PDZ domains and their protein partners that will require a re-evaluation of this domain's molecular function.http://www.imexconsortium.org) consortium through IntAct (PMID: 19850723) and assigned the identifier IM-14654][The protein interactions from this publication have been submitted to the IMEx ( C. elegans proteins assayed the interactions of 25 of the nematode's 62 PDZ domain proteins. Although these 25 proteins were found to be involved in 218 interactions, whether the different PDZ domains played a direct role was not addressed [Because of its biological importance, the PDZ  domain has been intensively studied at the structural and functional level. Proteins containing PDZ domains frequently serve as molecular scaffolds, which assemble signaling complexes needed for efficient and specific signal transduction at defined sub-cellular sites, such as at polarized epithelial cell junctions, or synapses in neurons . Early wddressed ,15. No cC. elegans. We generated a versatile resource, with each domain in the Gateway system, allowing facile transfer to different expression systems. As an example, we made a yeast array of the 93 PDZ domains and provide a proof-of-principle for its application to the discovery of PDZ domain targets for any protein or peptide of interest. In addition, from a separate yeast two-hybrid (Y2H) screen, we identified more than 650 potential partners for these domains. A large number of these interactions were independently validated using a co-immunoprecipitation approach. An analysis of these interactors implicates PDZ domains in a broad range of cellular functions. Unexpectedly, many of the interactions did not involve a C-terminal consensus sequence, suggesting that PDZ domains frequently bind their partners in a hitherto uncharacterized mode.Here, we describe the characterization and cloning of every single one of the 93 PDZ domains from C. elegans. Through an exhaustive cross-database search, we identified a total of 93 PDZ domains in 62 distinct proteins, not counting isoforms sharing domains , and thactome 8 , which hactome 8 ,19 for pPooling the results from the two screens, we obtained 674 interactions involving 469 proteins and 78 PDZ domains Figure . In manyIt is well established that some Y2H interactions do not reflect a physiologically relevant binding events between proteins. Ideally, these interactions need to be validated in vivo. Such tests are fastidious and not compatible with large-scale studies. We therefore sought to assay a subset of our Y2H interactions, using a very distinct experimental system, namely co-affinity immunoprecipitations (co-IP) from human 293T cells. First, we choose 13 different interactions, found in the cDNA screen, involving 11 PDZ domains and 9 interacting proteins. All these interactions were detected by co-IP using constructs encompassing the PDZ interacting full-length proteins, with their native C-terminus . There As a last example, both via our global screen and using the PDZ-domain array, we detected an interaction between MIG-5 and PRY-1. Previous studies had mapped the interaction between PRY-1 and MIG-5 to the N-terminal half of MIG-5 , which dC. elegans.It is clear that global Y2H screens only reveal a fraction of potential protein-protein interactions . Among oBy conducting a comprehensive, domain-centered interactome study, we have clearly illustrated at the genome scale the degree of promiscuity and discrimination that governs interactions between individual PDZ domains and their protein partners. This approach also revealed that PDZ domains frequently interact in a non-canonical fashion. This broadens our understanding of PDZ domains and should guide future functional studies.http://smart.embl-heidelberg.de) [Domain boundaries where obtained by cross searching Wormbase WS150  and SMARberg.de) . Each doPrimers, containing Gateway B1 and B2 recombination tails, were designed using the OSP program as described ,47 incluGAL1::HIS3 on minimal medium lacking leucine and histidine but containing 20 mM 3-amino-1,2,4-triazole (3-AT) in the absence of any AD-containing vector.DB-ORF plasmids were transformed into yeast strain MaV203 using standard transformation protocols . Auto-acC. elegans AD-wrmcDNA and AD-ORFeome1.0 libraries [6 colonies were screened for each bait strain tested with the AD-wrmcDNA library and a minimum of 1.5 × 105 colonies were screened for each bait strain tested with the AD-ORFeome library. After 4 to 5 days at 30°C, single 3-AT resistant colonies were picked on synthetic complete medium lacking leucine, tryptophan, and histidine and containing 20 mM 3-AT  and then rearrayed on fresh SC, Leu-, Trp-, His-, 20 mM 3-AT plates.Bait strains containing a single pDB-PDZ were individually transformed with the ibraries  as describraries . A minim using toothpicks, and lysed by incubating for 10 min. at 37°C and 10 min. at 95°C. For each PCR, 0.3 μl of lysis mix was used. AD inserts were amplified using primers 5'-CGCGTTTGGAATCACTACAGGG and 5'-GGAGACTTGACCAAACCTCTGGCG (AD and TERM respectively). DB inserts were amplified using primers 5'-GGCTTCAGTGGAGACTGATATGCCTC (DB) and TERM. PCR products were sequenced using the AD or DB primers.Colonies showing an activation of at least two of the three Y2H reporter genes were PCR amplified, as described above. PCR products showing a single band on ethidium bromide gel were sent for sequencing. The quality of the sequence obtained was determined as described  by movinGap repair was used to retest all Y2H interactions as described . When anMATα, ura3-52, his3-200, ade2-101, leu2-3, 112, gal4Δ, met-, gal80Δ, MEL1, URA3::GAL1UAS -GAL1TATA-lacZ). Individual ORFs of proteins of interest were cloned into DB vector (pGBT9) by Gateway recombinational cloning and the resulting constructs transformed into haploid AH109 yeast strain . Interactions between each PDZ and a given ORF was tested through mating of the two yeast strains. Phenotypic testing evaluated growth of diploid cells on selective medium , which is dependent in part upon the expression of the GAL1::HIS3 selective marker gene.All PDZ domains were transferred into AD vector (pACT2) by Gateway recombinational cloning and transfected into the haploid Y187 yeast strain . Cells were cultured for 48 hours in DMEM medium, and lysed in 0.1% NP-40 buffer ). Lysates were cleared by centrifugation at 14,000 × g and subjected to co-immunoprecipitations of protein complexes using anti-HA (clone12CA5) sepharose beads. Purified complexes and control lysate  samples were separated on Nu-PAGE Bis-Tris 4-12% gels (Invitrogen), and MYC and HA tagged proteins were detected using standard immunoblotting techniques. Antibodies used were mouse monoclonal anti-MYC  and monoclonal anti-HA .C. elegans most attributes are currently inferred from electronic annotation (IEA).The Textpresso database  was usedNL, JP, SB and SO carried out the experiments. NL performed the bioinformatic analysis. NL, JP and JR analyzed the data. JP, JPB and JR conceived the study. JR wrote the manuscript. All authors have read and approved the final manuscript.Definition of consensus classes. Additional file Click here for filePDZ domains cloning and annotations. Additional file Click here for filePDZome network. Additional file Click here for fileFunctional annotation of interacting proteins. Additional file Click here for fileVerification of two-hybrid interacting pairs by co-immunoprecipitation. Additional file Click here for fileCo-IP verification of yeast two hybrid interacting pairs identified in AD-wrmcDNA and AD-ORFeome screens using a B2 tailed construct. Additional file Click here for fileNegative controls of immunoprecipitations shown in Figure Click here for file