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Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast.

Large-scale changes (gross chromosomal rearrangements [GCRs]) are common in genomes, and are often associated with pathological disorders. We report here that a specific pair of nearby inverted repeats in budding yeast fuse to form a dicentric chromosome intermediate, which then rearranges to form a translocation and other GCRs. We next show that fusion of nearby inverted repeats is general; we found that many nearby inverted repeats that are present in the yeast genome also fuse, as does a pair of synthetically constructed inverted repeats. Fusion occurs between inverted repeats that are separated by several kilobases of DNA and share >20 base pairs of homology. Finally, we show that fusion of inverted repeats, surprisingly, does not require genes involved in double-strand break (DSB) repair or genes involved in other repeat recombination events. We therefore propose that fusion may occur by a DSB-independent, DNA replication-based mechanism (which we term "faulty template switching"). Fusion of nearby inverted repeats to form dicentrics may be a major cause of instability in yeast and in other organisms.

Large-scale chromosome changes (gross chromosomal rearrangements [GCRs]) occur in all genomes and can occur at a high frequency. Twin studies show frequent recombination between nearby (<1-Mb) large (>1-kb) direct repeat sequences (Bruder et al. 2008). Complex genome rearrangements cause loss or duplication of genes that affect human pathology (such as loss or gain of the PLP1 gene that affects myelination in humans) (Lee et al. 2007). Numerous large-scale changes are present in cancer cells and are thought to contribute to the cancer phenotype (Bignell et al. 2007). Bacterial genomes also suffer large-scale rearrangements (Tillier and Collins 2000). How these changes occur is largely unknown, and is of interest in understanding both pathology and genetic diversity.
The mechanisms underlying GCR formation appear complex, and may involve some combination of DNA replication, double-strand breaks (DSBs), repeat sequences, and unstable chromosome intermediates. One common view is that GCR formation is initiated by telomere or DNA replication dysfunction, leading to DSBs and subsequent fusion of two DSBs. DSB fusions may occur by any of several repair mechanisms (Wong et al. 2000;Kolodner et al. 2002;Murnane and Sabatier 2004;Weinstock et al. 2006), or the breaks may be capped by telomere addition (Chen and Kolodner 1999;Putnam et al. 2004;Sabatier et al. 2005; see Mizuno et al. 2009 for discussion). Recent studies of instability in yeast are in general consistent with the conclusion that DSBs can be intermediates in instability (Myung et al. 2001;Hackett and Greider 2003;Lemoine et al. 2005;Rattray et al. 2005;Narayanan et al. 2006). The question of whether GCRs also arise from an alternative replication-based mechanism, without a DSB intermediate, remains speculative. However, replication-based mechanisms have been inferred from complex rearrangements in human disorders (Lee et al. 2007) and rearrangements in bacteria (Bi and Liu 1996).
Repeat sequences present in genomes contribute to the formation of GCRs (whether a DSB is involved or not).
Immediately adjacent repeat sequences, called palindromes, may form hairpin structures that are easily broken and precipitate further genome rearrangements (Lobachev et al. 2002). Once a damaged structure (e.g., DSB or stalled fork) is formed, repeats may provide sequence homology that directs DNA repair (Zhou et al. 2001;Dujon 2006;Schmidt et al. 2006;Voineagu et al. 2008;Pennaneach and Kolodner 2009;Putnam et al. 2009). Repeats in eukaryotes are plentiful (Richard et al. 2008), and their recombination in mammalian cells is common (Zhou et al. 2001;Lobachev et al. 2007). Pairs of nearby repeats studied in this report are of special interest, since their close proximity and extensive homology may render them highly prone to rearrangements.
A third issue concerning GCR formation is whether they involve unstable chromosome intermediates. The presence of an unstable chromosome would readily explain how one cell generates progeny with many different chromosome structures, as commonly observed in cancer cells (Weinstock et al. 2006). An apparently common unstable chromosome is a dicentric, first described by McClintock (1941); once formed, the attempted segregation of the two centromeres of the dicentric to opposite poles during mitosis leads to chromosome breakage and further rearrangements (called the breakage-fusionbridge [BFB] cycle). Dicentrics and the BFB cycle have been invoked to explain the behavior of complex chromosome changes (Lo et al. 2002;Maser and DePinho 2002;Hackett and Greider 2003;Narayanan et al. 2006;).
In this study, we provide evidence that GCRs form when nearby inverted repeats fuse to form unstable dicentric intermediates. This study is an extension of a previous study describing a highly unstable region of a yeast chromosome (Chromosome VII [ChrVII]) that underwent multiple recombination events and formed unstable chromosomes, eventually forming a specific GCR (Fig. 1A,B; Admire et al. 2006). We reported that this highly unstable region contains inverted repeats and tRNA genes that appear to contribute to instabilityinstability that was further increased when DNA replication was disrupted. How the inverted repeats underwent recombination to form unstable chromosomes was unclear, and is clarified in this study. We now demonstrate that those inverted repeats first fuse to form a dicentric chromosome, which then rearranges further to form GCRs. We show that in budding yeast, fusion of nearby inverted repeats is a general phenomenon; we report that fusion occurs between naturally occurring and synthetically constructed inverted repeats present at different sites in the yeast genome. We then investigate the mechanism of fusion and find, surprisingly, that fusion does not require known DSB repair and replication fork rescue pathways, nor does it proceed by mechanisms known to mediate direct or inverted repeat recombination. We therefore suggest a replication-based mechanism of fusion of nearby inverted repeats. In an accompanying study, Mizuno et al. (2009) also present evidence for fusion of nearby inverted repeats in fission yeast, and Figure 1. Chromosome system to detect instability. (A) Two homologs of ChrVII and mutant alleles on each allow for genetic detection of chromosome changes. The CAN1 gene has been removed from ChrV and inserted into one copy of ChrVII. Selection for the loss of the CAN1 gene allows growth of cells with any of three types of chromosome changes, including simple loss, allelic recombinants, and mixed colonies. Mixed colonies contain cells of multiple genotypes, including a specific translocation. See the Materials and Methods for details. (B) Configuration of elements in the ChrVII 403 site and the geometry and order of how fusion may occur. Two tRNA genes (pentagons) transcribe toward the oncoming fork and slow replication. Fusion between the two LTR s repeats (S2 and S3), shown diagrammatically, forms a dicentric, followed by recombination between the two LTR d sequences (D7 and D11) to form the specific translocation.
suggest that fusion events also occur by a DSB-independent, replication-based mechanism.

Results
The genetic system that led us to the discovery of inverted repeat fusion is shown in Figure 1A (Carson and Hartwell 1985;Admire et al. 2006). Briefly, we constructed a haploid yeast strain that contains an extra copy of ChrVII (a disomic strain). Any changes to this extra chromosome should not compromise cell viability per se. The CAN1 gene, inserted near the left telomere of the extra chromosome, provides the means of identifying cells with chromosome changes. CAN1 encodes arginine permease that imports arginine as well as the toxic analog canavanine; only cells that lose the CAN1 gene can grow in media containing canavanine (forming Can R colonies). Using multiple genetic markers, we detected three types of chromosome changes in Can R colonies: wholechromosome loss, normal allelic recombination, and other complex rearrangements that give rise to colonies with a sectored appearance (which we refer to as ''mixed colonies'') ( Fig. 1A). (Point mutations in CAN1 occur more rarely [Weinert and Hartwell 1990.]) Mixed Can R colonies contain cells of multiple genotypes, whereas normal Can R colonies contain cells of a single genotype (see the Materials and Methods). Previously, we were able to infer that a mixed colony arises from a cell with an unstable chromosome, whereas a normal colony arises from a cell with a stable chromosome (Admire et al. 2006).
The initial focus of this study is to determine the nature of the unstable chromosome that forms mixed colonies. We used rad9 mutants here merely as a tool to study unstable chromosomes, because unstable chromosomes are formed more frequently in rad9 mutants than in wild-type (Rad + ) cells (Admire et al. 2006;this study). We emphasize that unstable chromosomes are formed in other mutants as well (some at an even higher frequency than in rad9 mutants) (see below).
A major clue to solving the structure of the unstable chromosome came from the structure of a specific translocation (the ''Delta 7/Delta 11'' [D7/D11] translocation) present only in mixed colonies (Fig. 1B). Our previous analyses suggested that this translocation probably arises by multiple deletion and fusion events centered at a site 403 kb from the left end ( Fig. 1B; Admire et al. 2006). This ChrVII 403 site contains long terminal repeat (LTR) sequences as well as tRNA genes that appear to contribute to instability (Admire et al. 2006;H Jones and T Weiner, unpubl.). LTRs are derived from retrotransposons (Kim et al. 1998) and are present in hundreds of copies in the yeast genome. (The Sigma 2 [S2] and Sigma 3 [S3] repeats are derived from Ty3 elements, are 278 and 245 bases long, share 82% identity, and are 3.3 kb apart, while D7 and D11 are from Ty1, are both 331 bases, share 97% identity, and are 133 kb apart.) Given the structure of the D7/D11 translocation, we suggest a relatively simple model in which one event fuses S2 and S3 inverted repeats to generate a dicentric chromosome, depicted in Figure 1B, and a second event fuses D7 and D11 direct repeats to resolve the dicentric and form the translocation. We first test the hypothesis that a dicentric intermediate is involved in ChrVII's instability.

A specific dicentric is present in cells that form mixed colonies
We employed a PCR assay to test for the presence of the specific dicentric ( Fig. 2A). We reasoned that the dicentric, if formed, might be present at a low but detectable amount in a culture of cells, and the amount of dicentric might predict the frequency of Can R mixed colonies arising from that culture. We therefore grew (in rich media) each of 10 single rad9 cells (initially containing an unrearranged ChrVII) into 10 independent cultures (first to colonies on plates, then each into liquid culture). We then performed two tests on each of these 10 cultures. We isolated genomic DNA from each culture and subjected it to amplification using primers that should detect the specific dicentric chromosome ( Fig. 2A). We did, in fact, detect a specific PCR fragment of the correct size and (B) Genomic DNA from unselected cultures of Rad + , rad9, and rad51 were subjected to PCR amplification using primers 1 and 2. Gels show qualitative PCR results. The rad9 cultures were also analyzed for their frequencies of mixed colonies and quantitative PCR to determine the amount of dicentric. Spearman correlation test was performed to correlate instability and dicentrics. Spearman rank correlation coefficient (r) is 0.973; P-value, <0.0001. (Lanes 4,8,9) Three of the Rad + cultures are faintly positive for the dicentric fragment. Primers to RSP5 were used as PCR controls. sequence if S2 and S3 inverted repeats fused to form the dicentric (see Supplemental Fig. S1 for sequence). Control PCR reactions failed to amplify the same DNA fragment from native ChrVII 403 site sequences (native sequences were made by PCR-amplifying the ChrVII 403 site) (data not shown). (We note that these PCR primers would amplify the same DNA fragment if it arose by an inversion event; a formal possibility that we eliminate by genetic and molecular tests [Supplemental Fig. S2; see below]).
We next tested whether the amount of the PCR product correlates with the frequency of mixed colonies in the 10 cultures. If so, cultures with quantitatively more PCR product should give rise to more mixed colonies. We determined the amount of dicentrics by quantitative PCR, and the frequency of mixed colonies by a quantitative genetic assay (see the Materials and Methods). We found that, in fact, cultures that had more dicentrics gave rise to a generally higher frequency of mixed colonies (correlation coefficient 0.97; P < 0.001) (Fig. 2B). The concentration of dicentrics varied between cultures, with an average of about one in 2000 rad9 cells (and one in 100,000 cells in Rad + cells) (Supplemental Fig. S3).
We further tested the correlation between the amount of PCR product and instability by studying strains with low instability (Rad + , wild-type cells) and high instability (rad51 mutants with a defect in homologous recombination [HR]) ( Fig. 2B; discussed further below). Again, we found more dicentric PCR product in rad51 mutants with high instability than in Rad + strains with low instability (Fig. 2B). We observe this correlation in other mutants as well (see below). We conclude that the specific dicentric depicted in Figures 1B and 2A is most likely a key intermediate to instability and to formation of the specific D7/D11 translocation shown in Figure 1B.

Genetic test confirms that dicentrics cause the instability in mixed colonies
Although results from the PCR assay described above argue strongly that a specific dicentric causes instability, we cannot directly detect intact dicentric chromosomes (due to their fragility and low abundance). We therefore turned to a genetic approach to test if dicentrics are, in fact, intermediates in instability.
Our genetic test uses a temperature-sensitive mutant of a kinetochore protein, ctf13-30, that functions properly at 23°C but not at 36°C (Doheny et al. 1993). Ctf13 connects microtubules to the kinetochore. The idea of using this mutant is as follows: When a dicentric undergoes chromosome segregation during mitosis and Ctf13 is active, the DNA will break, but if Ctf13 is inactive, the microtubule-kinetochore connection will break instead of the DNA (Fig. 3A). (DNA breakage is required to form mixed colonies, since mixed colonies contain cells of multiple genotypes.) We therefore predicted that Ctf13 status will affect the frequency of mixed colonies if dicentrics are intermediates, whereas Ctf13 status will not affect the frequency of mixed colonies if dicentrics are not intermediates. To test this prediction, we grew rad9 and rad9 ctf13-30 cells at the permissive (23°C) or the restrictive (36°C) temperature, and determined the level of dicentrics by quantitative PCR and the level of instability from the frequency of mixed colonies (Fig. 3B). (We found that the status of Ctf13 did not drastically affect the amount of dicentrics formed, as measured by quantitative PCR; there is a fourfold increase in dicentrics in Ctf13-defective cells.) We found that Ctf13 status did, in fact, affect instability; rad9 ctf13-30 mutants had an eightfold lower frequency of mixed colonies when grown at a restrictive temperature compared with the permissive temperature, and compared with rad9 CTF13 + strains grown at either temperature. Thus, the amount of dicentric formed was largely independent of Ctf13 activity (there is actually more dicentric in Ctf13-defective cells), but the fate of the dicentrics was dependent on Ctf13 status (fewer mixed colonies in Ctf13-defective cells). This Ctf13 result is consistent with the hypothesis that the specific dicentric detected by PCR is an intermediate to instability.
We note that rad9 ctf13-30 cells have a higher frequency of chromosome loss and of allelic recombination when grown at the restrictive temperature compared with the permissive temperature, as reported previously ( Fig. 3B; Doheny et al. 1993). An increase in chromosome loss was expected from the model; dicentrics missegregate and are lost when the Ctf13 kinetochore connection breaks. The higher frequency of allelic recombination in ctf13-defective cells has also been reported for monocentric chromosomes in both ctf13-defective cells (Doheny et al. 1993) and for cells treated with the microtubule inhibitor benomyl (Wood and Hartwell 1982). The cause of increased allelic recombination in any of these studies is unknown, but it is unlikely that it is due to dicentric behavior (see the Discussion; Supplemental Fig. S4).
In sum, we conclude that inverted repeats in the ChrVII 403 site fuse to form a dicentric that causes the instability evident in mixed colonies. We next show that fusion of nearby inverted repeats is a general phenomenon in the yeast genome.

The pairwise fusion of many nearby inverted repeats in the yeast genome
To test if inverted repeat fusion is general, we again used a PCR-based assay. By examining the yeast genome, we found that there are ;15 sites in budding yeast that contain LTR inverted repeats flanked by tRNA genes, a geometry similar to that of the unstable ChrVII 403 site (Fig.  4A). We chose five of these sites to test for their fusion of inverted repeats. We isolated genomic DNA from cells and subjected it to PCR amplification using appropriate primers (as in Fig. 2A). We developed separate sets of primers that could detect either acentrics or dicentrics, since theoretically either is equally possible (see Fig. 4D). We . Inverted repeats at five sites in the yeast genome fuse to form dicentrics and/or acentrics. (A) The sites screened for fusion contain tRNA genes (pentagons), repeat sequences (gray and brown arrows are involved in the fusion reaction, whereas clear ones are not), and, in one case, an origin (square). Inverted repeat fusion forms either dicentric or acentric fragments using primers in schemes shown in C. (B) DNA fragments are detected at all five sites tested, using appropriate primers specific for each site. Primer sequences are in Supplemental Figure S9. Analysis of three sites yielded both acentric and dicentric fragments. Asterisks indicate where DNA fragments should have appeared, if they formed. The DNA sequence of all of the DNA fragments has been confirmed (Supplemental Fig. S5). (C) If the fusion junctions for acentric and dicentric chromosomes are identical, then they could have arisen by a symmetrical mechanism. This mechanism can conceptually be viewed as a crossover depicted here; one event generates both acentrics and dicentrics. (Not shown here are stalled forks as intermediates; this diagram is meant to convey the idea of a symmetrical event only.) (D) On the other hand, if the fusion junctions for acentric and dicentric chromosomes are not identical, then we infer that acentrics and dicentrics must be formed by separate events. In this case, the DNA polymerase is ''jumping'' between different sequences to form acentrics than it does to form dicentrics. Note that symmetric joints can also be formed by an asymmetrical mechanism, if the DNA polymerases for the acentric and dicentric ''jump'' between identical sequences. detected DNA fragments diagnostic of acentric and/or dicentric chromosomes at all five genomic sites tested (Fig. 4B). To get an idea of how frequently these other nearby inverted repeats fuse, we performed quantitative PCR on two additional sites (ChrVI 160 and ChrX 542 , in addition to the ChrVII 403 ), and found that the frequency of fusion events in Rad + strains at all three sites was comparable (1.5 3 10 À5 , 4.7 3 10 À5 , and 8.1 3 10 À5 for the three sites, respectively) (see Supplemental Fig. S5I).
By examining the DNA sequence of the fused pairs of inverted repeats, we can deduce some features of the mechanism(s) of fusion. First, we found that fusion always involves some sequence homology; fusion can occur between two sequences that share as little as 20 base pairs (bp) of homology with a 1 bp mismatch (ChrV 137a site) (Supplemental Fig. S5D), or as much as 148 bases of exact homology (ChrVII 403 dicentric) (summarized in Supplemental Fig. S5H). Genetic tests described below are consistent with the involvement of only limited homology in the fusion reactions.
Second, we found that fusion of inverted repeats can happen between repeats that are as far as 5.4 kb away from each other (ChrX 542 site) (Supplemental Fig. S5H). Since this site had the largest distance between the two repeats, and still readily formed both dicentric and acentric chromosomes, it is likely that the maximum distance for fusion is larger (Supplemental Fig. S5I). Distance requirements for fusion will be the focus of a future study.
Third, we found evidence that bears on the issue of whether one event generates both an acentric and dicentric chromosome (Fig. 4C), or whether one event generates either an acentric or a dicentric chromosome but not both (Fig. 4D). We term these two options ''symmetric'' and ''asymmetric'' events, respectively. The evidence that bears on this question comes from the sequence of the fusion junctions for sites where both acentric and dicentric chromosomes are formed. Surprisingly, we found that, for two of them (ChrVII 403 and ChrV 137b ), the fusion junction in the acentric product was different from the dicentric product (using different bases within the repeats as depicted in Fig. 4D; actual sequences in Supplemental Figs. S1, S5). For the ChrVII 403 site, we found that 16 of 16 acentrics used the same sequence (within 8 bases of homology between S2 and S3), whereas 13 of 13 dicentrics analyzed used a different sequence (within 150 bases of homology between S2 and S3). For the third site (ChrX 542 ), the fusion to form acentrics and dicentrics does occur in the same sequence (as in Fig. 4C), so there is a formal possibility that, for this site, both acentrics and dicentrics are generated by a single event. Formation of acentric and dicentric chromosomes by an asymmetric event has mechanistic implications that are addressed in the Discussion.

Synthetic inverted repeats also fuse to form dicentrics associated with instability
To further test the generality of the fusion reaction, we generated a synthetic inverted repeat consisting of non-LTR sequences (all of the events analyzed thus far contain LTR sequences). This synthetic inverted repeat also does not contain any tRNA genes that likely contribute to instability. We divided the URA3 gene into three fragments, with each pair of consecutive fragments sharing ;200 bp of sequence homology (designated as ''UR,'' ''RA,'' and ''A3'') ( Fig. 5A; Supplemental Fig. S6). We first inserted the URA3 modules into the ChrVII sites that form the dicentric and D7/D11 translocation (the 403 and 535 sites, respectively) (see Figs. 1B, 5A). We allowed instability of these genetically modified cells to occur, identified mixed colonies by selection for Can R , and analyzed cells in the mixed colonies for URA3-mediated events. Overall, we found that the URA3-mediated events were virtually identical to the LTR-mediated events. First, we found that the URA3 sequence-containing inverted repeats formed dicentric chromosomes, just as the LTR sequences S2 and S3 had (Fig. 5B). Second, the URA3 dicentric intermediate was frequently resolved to form a translocation (Supplemental Fig. S7), just as with the LTR-containing dicentric. As discussed further below, we also found that the genetic requirements for fusion of the URA3-based inverted repeats is the same as for fusion of the S2 and S3 repeats (neither reaction requires the RAD52 or RAD59 genes) ( Fig. 5B; see below).
We also used the synthetic inverted repeat module to test whether fusion is for some reason constrained to occur in the ChrVII 403 site. We inserted the synthetic inverted repeat module into two additional ChrVII sites (the ChrVII 110 and ChrVII 461 sites) that do not contain LTR fragments, do not contain tRNA genes, and are in completely different regions of the chromosome (one 110 kb from the telomere, and the other 37 kb from the centromere). We were able to identify URA3-containing dicentrics formed at both sites (Fig. 5B), confirming that nearby inverted repeat fusion is a general phenomenon. These studies show that fusion of nearby inverted repeats is not restricted to types of DNA sequences, to sequences near tRNA genes, or to specific regions of a chromosome. (We also identified Ura + translocations, formed by resolution of the dicentric, from Ura modules inserted at the ChrVII 461 site but not the ChrVII 110 site, a result for which we have no explanation).
Finally, we used the synthetic inverted repeat module to re-examine how much sequence homology is required to form dicentrics. We found that 60 bases of homology, but not 20 or 0, allowed dicentric formation (Fig. 5B). In sum, the lengths of homology involved in LTR and URA3 inverted repeat fusion are consistent with homologies of 60 bases and greater and, in some cases, as few as 20 bp undergoing fusion.
Major DNA repair and replication fork pathways and the mechanism of inverted repeat fusion We next used genetic analyses to address questions of mechanism: How does the cell normally prevent such a deleterious fusion reaction, and, when not prevented, how does the fusion reaction then occur? We first considered the possibility that fusion of inverted repeats occurs by the same mechanisms by which other types of repeats recombine. The mechanisms of recombination between direct repeats (Klein 1988;Thomas and Rothstein 1989), between inverted repeats (Bai and Symington 1996;Jinks-Robertson et al. 1993;Fasullo et al. 2001) and between sister chromosomes (Paulovich et al. 1999, Fasullo et al. 2001) has been thoroughly studied in budding yeast. The key observations in those studies are that recombination is decreased >100-fold by mutations in either RAD52 or RAD52 and RAD1. Rad52 is a single-strand annealing (SSA) protein required for most HR reactions (Krogh and Symington 2004), and Rad1 is a nuclease with roles in HR and nucleotide excision repair. We therefore determined the frequency of dicentrics and instability at the ChrVII 403 site in rad52, rad1, and rad52 rad1 mutants. Strikingly, we found that inverted repeat fusions were actually increased (>10-fold) in rad52 and rad52 rad1 double mutants compared with wild-type (Rad + ) cells (determined from dicentric PCR or mixed colonies) ( Table 1). (Some of the fusion events that occurred in rad52 mutants may be Rad1-dependent, since the level of dicentric in rad52 rad1 double mutants was twofold lower by quantitative PCR [ Table 1]; this does not change our overall conclusion that the inverted repeat fusion occurs by a RAD52, RAD1-independent mechanism. We also found that rad52 and rad52 rad1 also gave rise to the specific D7/D11 translocation, a product formed when the dicentric intermediate is resolved). Other mutants shown in other studies to decrease inverted repeat inversions (e.g., rad51 and rad59) (Bai and Symington 1996) also lead in our study to an increase in inverted repeat fusion at the ChrVII 403 site (Table 1). We also analyzed the roles of RAD52 and RAD1 in fusion at two other sites-ChrVI 160 and ChrX 542 -by using quantitative PCR. We found that fusion between inverted repeats occurred efficiently in rad52 and rad52 rad1 mutants; fusion products are present at nearly the levels formed in wild-type cells (Supplemental Fig. S5I). We do not know why the ChrVII 403 site is more unstable in a rad52 mutant than the other two sites. The ChrVII 403 site is at a replication terminus, and that might contribute to its instability and different genetic regulation (Admire et al. 2006). We therefore conclude that inverted repeat fusion occurs by a mechanism that is very different from ''conventional'' recombination between inverted and direct repeats (forming inversions or deletions, respectively).
To further address the mechanisms that underlie inverted repeat fusion, we focused on the observation that fusions are increased when DNA replication forks stall (either by lowering dNTPs, by defects in a DNA helicase, or by tRNA gene transcription) (Admire et al. 2006;H Jones and T Weiner, unpubl.). We therefore suggest that inverted repeats fuse by a mechanism related to stalled replication forks and perhaps to how they recover. To guide our studies, we provided a working model of replication fork biology ( Fig. 6; Atkinson and McGlynn 2009;Branzei and Foiani 2009). A stalled replication fork (Fig. 6, top) may resume replication (data not shown), undergo a DSB (Fig. 6, left), or undergo sister strand annealing reactions to form a regressed fork (Fig. 6, center) or a hemicatenane (Fig. 6, right). Some outcomes from these structures are shown, and others are possible  Fig. S6 for details of module construction and chromosome insertion). The two modules were then inserted into the specific sites in ChrVII as indicated. Inverted repeat recombination joins UR to RA modules via recombination of shared ''R'' sequences to form a dicentric. A second recombination event joins ''A'' to ''A3'' to form an intact URA3 gene, rendering the cells Ura + . (B) Each rad9 strain contains a UR or RA module in one of three sites on the left arm of ChrVII, and all strains contain the A3 module at one site on the right arm. The UR modules differ in the amount of sequence homology shared with the RA module (size of ''R'' is 200, 60, 20, or 0 bp of homology, as indicated). Each modified rad9 strain was analyzed for the presence of the specific PCR fragment diagnostic of a dicentric chromosome, in four independent cultures, using dicentric URA primers (Supplemental Fig. S9). Can R mixed colonies were generated from each rad9 strain, and the frequency of Ura + cells in cells from mixed colonies was determined (see the Materials and Methods). (**) The frequency of Ura + colonies in Can s cells is shown in Supplemental Figure S8. as well (e.g., the regressed fork may be cleaved by a Holliday junction resolvase to form a DSB) (Atkinson and McGlynn 2009).
To begin to test if any of these pathways are relevant to the fusion of inverted repeats to form dicentrics, we measured the frequency of mixed colony formation in a number of mutants thought to have roles in replication fork recovery. The frequency of mixed colonies is a suitable quantitative surrogate for the frequency of dicentric formation (see Fig. 2), and for some mutants we also performed quantitative dicentric PCR (Table 1). Our genetic and molecular analyses uncovered several relationships between replication fork recovery pathways and nearby inverted repeat fusion. First, as noted above, we found that the Rad52 protein was not required for dicentric formation; fusion of inverted repeats is Rad52-independent. Second, we found that defects in other end-joining pathways-such as nonhomologous end-joining (NHEJ), microhomology-mediated end-joining (MMEJ), and SSA-had little effect on instability as single mutants (Ku70, Lig4, and Rad1; NHEJ/ MMEJ, SSA) (Paques and Haber 1999). In strains where events occur more frequently (i.e., rad9 mutants), these events do not require any of these genes (i.e., rad9 and rad9 lig4 mutants have similar phenotypes). Inverted repeat fusion also does not require either Rad50, a component of the MRX complex that is required for both NHEJ and MMEJ, or Mus81, a prominent nuclease. Indeed, fusion events increase ;30-fold in both of these mutants, implying that they have a prominent role in the prevention of fusion events. These results suggest that fusion of inverted repeats coming from stalled forks has little to do with NHEJ, MMEJ, or SSA repair pathways.

Rad59
In addition to Rad51, the SSA protein Rad59 is thought to define a second distinct Rad52-dependent HR pathway that mediates annealing between sequences with shorter regions of homology (Spell and Jinks-Robertson 2003;Pannunzio et al. 2008). We found that strains lacking Rad59 showed an increase in mixed colony formation, and readily formed both dicentric and acentric chromosomes (Table 1; Supplemental Fig. S8); therefore, Rad59 acts like Rad52 and Rad51 to normally prevent inverted repeat fusion. We did make one surprising observation studying Rad59 that bears on how the dicentric is resolved. We found that Rad59 is required for resolution of the dicentric (involving recombination between D7 and D11 to from the D7/D11 translocation); neither rad59 nor rad9 rad59 double mutants generated this translocation (Table 1), while both mutants generated dicentric chromosomes. Events associated with the URA3 synthetic module also obey the same genetics as LTR recombination in all respects: The ''URA''-containing inverted repeats fuse to form the dicentric in the absence of RAD59, but the resolution of the dicentric to form a URA3-containing translocation is reduced 200-fold in rad9 rad59 mutants (Fig. 5B, cf. rad9 and rad9 rad59).

Pol32 and break-induced replication (BIR)-mediated events
There is increasing evidence that links replication stress to microhomology-mediated fusion events (for review, see Hastings et al. 2009a). A mechanism termed microhomology-mediated BIR (MMBIR) has been proposed to resolve stalled forks by mediating fusion events (for review, see Hastings et al. 2009b). Both homology-driven and microhomology-driven BIR require a nonessential subunit of DNA polymerase d, Pol32 (Lydeard et al. 2007;Payen et al. 2008). We thus tested if Pol32 was required for inverted repeat fusion, and for resolution of the dicentric to form the translocation. We found that instability is actually increased in pol32 mutants, and in rad9 pol32 double mutants when compared with Rad + and rad9 strains. We therefore infer that neither Pol32 nor BIR events are involved in either fusion of inverted repeats to form the dicentric or in resolution of the dicentric to form the D7/D11 translocation. Figure 6. A model of the pathways that may act on stalled replication forks. DNA polymerase stalls at a lesion (triangle). The stalled fork may undergo a DSB (i), or sister strand annealing, template switch events forming a regressed fork (ii) or hemicatenane (iii). DSBs may undergo DNA rejoining by NHEJ, MMEJ, HR, SSA, or telomere addition (not shown). Some genes tested in this study that may regulate each pathway are shown.

Conclusion of genetic analysis of inverted repeat fusion
In conclusion, our genetic studies first indicate that inverted repeat fusion occurs by a mechanism distinct from repeat recombination events reported previously in budding yeast. Second, fusion is prevented by genes proposed to act in replication fork recovery to form hemicatenanes (RAD51, RAD52, and RAD18). Third, fusion does not require genes that are involved in DSB repair, BIR, or hemicatenane formation (RAD1, RAD50, RAD51, RAD59, RAD52, yKU80, LIG4, POL32, and RAD18). These genetic results rule out several models, and lead us to speculate on a replication-based model addressed in the Discussion.

Discussion
In this study, we report and discuss below two major findings. First, we found that nearby inverted repeats fuse to form either dicentric or acentric chromosomes, and that dicentric formation is linked to chromosome instability. This fusion is general; it happens at multiple sites in the yeast genome, and at a synthetic site placed at three different locations on a single chromosome. Second, fusion of nearby inverted repeats is suppressed by many DNA repair pathways, and when it does occur, fusion is surprisingly independent of DSB repair and other major repair pathways. These and other results lead to a replication-based model of how fusions occur.

Inverted repeat instability in budding yeast
Inverted repeats represent potential sites of instability in both prokaryotic and eukaryotic genomes. Here, we found that nearby inverted repeats, consisting of either LTRs or synthetic inverted repeats, fuse to form acentric or dicentric chromosomes. (This reaction is mechanistically very different from an inversion between inverted repeats, so we used the phrase ''inverted repeat fusion.'') A search of the literature indicates that nearby inverted repeat fusion is an event that occurs in bacteria and in fission yeast as well (see below). The extent to which three nearby inverted repeats destabilize the budding yeast genome is not yet clear, and will depend in part on defining sequence parameters required for fusionparameters that may involve chromosome topology as well. We did find that the fusion events in budding yeast occur between inverted repeats with as few as ;20 bases of homology and separated by ;1.3-5.5 kb of DNA (Supplemental Fig. S5I). The frequency of inverted repeat fusion is about one in 100,000 wild-type cells at the ChrVII 403 site to eight in 100,000 at the ChrX 542 site. The inverted repeats in ChrVII 403 do appear to cause most of that chromosome's instability (H Jones and T Weinert, unpubl.); other inverted repeats on ChrVII probably also cause instability, though they have not yet been tested. Sites of inverted repeats also appear to be sites of translocation events that led to speciation of Saccharomyces species (Admire et al. 2006; AL Paek and T Weinert, unpubl.).

Inverted repeat fusion and instability in other genomes
After we identified inverted repeat fusion in budding yeast, we examined the literature seeking similar observations in other organisms. Inverted repeat fusions similar to that described in this study have been reported in two studies in bacteria and two in fission yeast. In particular, in one bacterial study, nearby inverted repeats present on a plasmid were found to undergo a complex rearrangement that did not involve RecA (Rad51 ortholog) (Bi and Liu 1996), and in a second study, DNA intermediates were identified that also appeared to be undergoing inverted repeat fusion (Ahmed and Podemski 1998). The geometry and RecA independence of these reactions suggest to us that they may occur by a similar mechanism as the events in budding yeast (see below for discussion of mechanism). Finally a third study of instability in fission yeast also appears due to nearby inverted repeat fusion. Albrecht et al. (2000) selected for the amplification of Sod2 that confers resistance to lithium chloride, and isolated strains that contained high copies of acentric chromosomes. Examination of the acentric suggests it was formed by fusion of two nearby inverted repeats. Finally, an accompanying study by Mizuno et al. (2009) also shows that nearby inverted repeats can form acentric and dicentric chromosomes in fission yeast.

Similarities and differences in instability associated with palindromes and nearby inverted repeats
Palindromes are a class of inverted repeat sequences separated by very few base pairs of DNA. Nearby inverted repeats can be separated by many kilobases of DNA. Both of these classes of repeat sequences lead to formation of dicentric and acentric chromosomes, but how they do so appears to differ. In budding yeast, Lobachev and colleagues (Narayanan et al. 2006;Voineagu et al. 2008) found that palindromes formed a hairpin or cruciform structure that rearranges to form acentric and dicentric chromosomes. Similarly, Lemoine et al. (2005) also found that palindromes cause instability, presumably also by formation of a cruciform. In Schizosaccharomyces pombe, 80-bp perfect inverted repeats form hairpin and cruciform structures and confer mitotic instability (Farah et al. 2002). Due to their unstable nature, palindromes are underrepresented in the human genome (Lobachev et al. 2000). Although the nearby inverted repeat fusion events reported here also lead to the formation of dicentric and acentric chromosomes, they must do so by a different mechanism, since the repeats are separated by many kilobases of DNA and hence are unlikely to form hairpin or cruciform structures (Lobachev et al. 2000).

A replication-based mechanism for fusion of nearby inverted repeats
The mechanism of inverted repeat fusion remains speculative. What we do know is that many repair and checkpoint control pathways normally suppress fusion of nearby inverted repeats ( Fig. 6; Table 1; Admire et al. 2006). It is also clear that fusions occur by a mechanism very different from those mediating recombination between inverted and direct repeats-events that can be stimulated by DSBs (Sugawara and Haber 1992;Bai and Symington 1996;Fasullo et al. 2001). Whatever the mechanism of inverted repeat fusion is, it should account for (1) the geometry of the fusion reaction, (2) the lack of involvement of Rad52 and other repair proteins, (3) the increased fusion of inverted repeats upon disruption of DNA replication, and (4) an asymmetric mechanism of fusion.
To account for these observations, we suggest a replication-based mechanism we call ''faulty template switching'' (Fig. 7). This mechanism bears a similarity to mechanisms proposed from studies in bacteria (Goldfless et al. 2006;Long and Kreuzer 2008;Atkinson and McGlynn 2009). In this model, a stalled fork fails to be rescued by error-free pathways (the Rad51, Rad18dependent hemicatenane pathway, for example) (see Fig.  6), and undergoes a different template switch event (fork regression, replication, and reversal) (Fig. 6) to form a socalled ''chicken foot'' structure. We suggest that, during fork restart, the nascent DNA strand may either undergo fork reversal (data not shown), pair with the correct sequence and be resolved by an error-free mechanism (as summarized by Atkinson and McGlynn 2009), or pair with an incorrect, nearby sequence (faulty template switch). This chicken foot-based faulty template switch reaction appears to be Rad18-independent. (Rad18 has been proposed to be involved in hemicatenane-based template switch [Fig. 6;Branzei et al. 2008].) How the pairing reaction occurs is unclear, since strand annealing proteins (i.e., Rad52) are not required. A speculative strand annealing reaction without Rad52 has been discussed recently (Hastings et al. 2009b). Once strand annealing has occurred, completion of DNA replication leads fairly directly to formation of a dicentric (or acentric) chromosome.
Although speculative, this model illustrates how a relatively simple error in fork restart (selection of the wrong sequence) can lead to a chromosome change. The model makes specific testable predictions; we hypothesize that faulty template switching requires a 59-39 exonuclease for degradation of the regressed fork, and an endonuclease to resolve the structure after annealing to a nonallelic sequence. Further work linking gene function to DNA structure will allow us to further test how inverted repeats fuse. Models having a DSB as an intermediate are also plausible.
Other examples of possible template switching Models involving some form of template switching have been proposed previously. In bacteria, a faulty template switch model was proposed to explain rearrangements between direct repeats (Goldfless et al. 2006). In that study, rearrangements were independent of RecA (a Rad51 ortholog), as inverted repeat fusion events reported here were. (In the bacterial study, rearrangements required a gene [DnaK] known to interact with DNA polymerases.) Fusion of nearby inverted repeats in bacteria (mentioned above) also suggest a template switch mechanism (Bi and Liu 1996;Ahmed and Podemski 1998). In yeast, template switching can occur during BIR, and is thought to be due to the unstable nature of replication forks during BIR (Lydeard et al. 2007;Smith et al. 2007Smith et al. , 2009 . Smith et al. (2007. Smith et al. ( , 2009 showed switching between allelic and nonallelic sequences, though those events require proteins involved in HR. Finally, a template switch reaction has been suggested to explain complex rearrangements in a human disorder (Lee et al. 2007). The template switch mechanism Lee et al. (2007) propose involves fusion between sequences sharing only very short stretches of sequence homology, seemingly an event that would not Figure 7. DNA replication-based mechanism of inverted repeat fusion. The top shows a chromosome region that contains inverted repeat sequences (red box, S2; blue box, S3), separated by several kilobases of DNA. The right fork stalls (not shown), then regresses to form the ''chicken foot'' structure with some S3 sequence at the 39 end of the regressed strand. On the right, the regressed fork normally reinvades at the appropriate S3 sequence, and forms a Holliday structure that then is cleaved and resolved to restore a fork (after Atkinson and McGlynn 2009). On the left, the regressed fork reinvades at the wrong S2 sequence. Then, the unpaired regressed strand (dark green) is degraded (pacman), and the invaded strand begins to replicate using the blue strand as a template (displacing the blue strand from the red strand and allowing annealing of light green to red, forming the S2-S3 joint; dotted line). Upon completion of replication, ssDNA breaks must separate the left red and blue strands and the gap is filled (thin red arc, details not shown), forming the dicentric with hybrid S2-S3 repeat and one intact S2 repeat. The dicentric may then undergo further instability. involve strand annealing activities (Hastings et al. 2009b). The extent to which a faulty template switch model between nonallelic sequences might explain rearrangements in budding yeast remains to be determined (Myung et al. 2001;Payen et al. 2008).
Clearly, understanding the mechanism of template switching is a technical challenge, in part given how infrequently these events occur. Recently, Mizuno et al. (2009) described a system that may permit dissection of replication fork stalling and template switching. Their results bear similarities to those reported here in two aspects: They found that replication forks stall in nearby inverted repeats, and that stalling causes the fusion of those inverted repeats to form acentric and dicentric chromosomes. In addition, Mizuno et al. (2009) failed to detect any DSBs as intermediates to fusion, and they suggested a template switch model with some similarities to that shown in Figure 7. However, the genetic requirements of those events in fission yeast are similar in some aspects (events are independent of Lig4 and Ku70) but differ in others (85% of the events require genes involved in HR); thus, the mechanistic similarities are as yet unclear. The role of genes in HR lead Mizuno et al. (2009) to propose that, following a template switch, a Holliday structure is formed and resolved to generate symmetric products. We do not have an explanation for the differences, though it is possible that initiation of faulty template switch events is similar, but resolution of intermediates may differ.

The role of Rad59
We also report here the surprising finding that Rad59 has a role clearly independent of Rad52. Specifically, we found that resolution of the dicentric to the D7/D11 translocation required Rad59, but not Rad52 or Rad51 (see Fig. 1B; Table 1). This result was surprising, because previous genetic analyses suggested that Rad52 is required for Rad59 function (Bai and Symington 1996;Davis and Symington 2001). However, recently, Pannunzio et al. (2008) found that chromosomal rearrangements in rad52 mutants were partially dependent on Rad59. Several other studies have shown that dicentric chromosomes are often resolved by translocations involving repetitive elements (Haber and Thorburn 1984;Surosky and Tye 1985;Pennaneach and Kolodner 2009). It is not clear whether these translocations require Rad59. The Rad59-dependent resolution of the dicentric chromosome will be the focus of a further study.

Broader implications for genome stability in mammalian genomes
It is clear that many eukaryotic genomes harbor nearby inverted repeats that might destabilize their chromosomes. The human genome contains a high frequency of large inverted repeats with high sequence homology (Warburton et al. 2004). Wang and Leung (2009) estimated the frequency of inverted repeats of >30 bp in length to be around one in 10,000 bp in the human genome. The extent to which inverted repeats fuse or cause instability in mammalian genomes is unknown. A recent study by Lange et al. (2009) found that fusion of large inverted repeats (0.3-to 2.9-Mb arms separated by 2.1-169 kb) on the human Y chromosome leads to the formation of dicentric chromosomes. Patients that harbored dicentric chromosomes exhibited spermatogenic failure, and in some cases Turner syndrome, a sex disorder that is characterized by ''XO'' mosaicism. Lange et al. (2009) hypothesize that the ''XO'' mosaicism seen in at least some of these patients is due to an unstable dicentric Y chromosome. Interestingly it has been shown recently that individuals with Turner syndrome have a higher incidence of cancer than the general population (Schoemaker et al. 2008). It is not yet known whether the increased cancer risk is in fact due to the existence of an unstable dicentric chromosome. Also, the extent to which these fusion events are caused by a faulty template switching mechanism, as opposed to a crossover event between sister chromatids, is unclear.

Yeast strains
Yeast strains are derivatives of the A364a strain described in previous studies and references therein (Weinert and Hartwell 1990), and are derived from TY200 or TY206 by transformation. TY200 is a ChrVII disome generated by T. Formosa, M. Carson, and L. Hartwell that is MATa +/hxk2TCAN1 lys5/+ cyh r /CYH s trp5/+ leu1/+ centromere ade6/+ +/ade3, ura3-52. TY206 harbors a rad9-null mutation, rad9Tura3, described by Weinert and Hartwell (1990). Derivatives were made by transformation (Gietz and Woods 2001) of appropriate DNA fragments, and genotypes were verified by Southern analysis, PCR, and/or phenotype. Mutant DNA fragments were generated by PCR amplification of KanMX4-marked mutations from the Euroscarf strain using primers that flank each gene. Candidate mutants were analyzed by PCR using outside primers, and restriction digests were performed in cases where mutant and wild-type alleles had similar-sized fragments. Genotyping for all strains was unambiguous. In some cases, we introduced a URA3 allele to replace the KanMX4 allele. The rad1 mutation was generated by PCR-amplifying the hygromycin gene from pAG32 with primers that included 45 bp of homology with DNA flanking the rad1 ORF. rad9 ctf13-30 alleles were generated by a ''pop-in-popout'' strategy. TY206 was transformed with HindIII-linearized pBFS218 ctf13-30 (gift of F. Spencer) under selection for Ura + . We then isolated 5-Fluoroorotic acid-resistant derivatives (''popouts'') that were screened for temperature sensitivity at 37°C. Further details of strain constructions are available upon request.
The URA3 inverted repeat module was constructed as shown in Supplemental Figure S5. Briefly, fragments of the URA3 gene were joined to either the Nat1 gene (RA:NatR:RU cassette) or to KanMX4 (A3:KanMX4). These cassettes were inserted into plasmids that contain ;500 bp of sequence-flanking sites 110, 403, 461, or 535. Each module was inserted into ChrVII by restriction digestion and transformed into cells, and inserts were selected by growth on drug-containing plates. Candidate insertions were confirmed by genetic analyses. For example, the rad9 430:RA:NatR:RU cassette is linked to the CAN1 gene, and resides genetically between TRP5 and CYH2, mapped by analysis of allelic recombinants. Canavanine (Can; 60 mg/mL), geneticin (100 or 500 mg/mL), hygromycin B (300 mg/mL), and nourseothricin (Nat; 50 mg/mL) were used at the concentrations given.

Chromosome instability assays
Genetic analysis of ChrVII instability was performed as described in Admire et al. (2006). Briefly, we grew cells on synthetic media lacking adenine (Ade) to ensure selection for both ChrVII homologs. We then plated cells on rich media (YEPD, 2% dextrose) and, after 2-3 d of growth at 30°C, identified single colonies that contained 1 3 10 6 to 3 3 10 6 cells. Synthetic media were as described (Sherman et al. 1986). To identify the types of chromosome changes that occurred as cells grew on rich media, we resuspended individual colonies in water, counted, and plated on selective media consisting of either synthetic media supplemented with 60 mg/mL Can and all essential amino acids except arginine and serine, or the same selective media also lacking Ade. Cells plated on selective media containing Ade identify chromosome loss events as colonies that prove to be Can R Ade À after replica plating on media lacking Ade (see Fig. 1A). Cells plated on selective media without Ade identified allelic recombinants and mixed colonies (both Can R Ade + ). We scored the morphology of colonies as either ''round,'' which generally proved to be stable allelic recombinants, or ''sectored/mixed,'' which generally proved to contain cells of many different genotypes. We performed ''lineage assays'' to confirm that cells in allelic recombinants contained the same genotype (typically >95% of the cells had the same genotype), and that cells in mixed colonies contained multiple genotypes (see Admire et al. 2006), including chromosome loss and allelic recombinants in multiple genetic intervals shown in Figure 1A. In all strains, we verified that round and mixed colonies contained single or multiple genotypes, and corrected the frequencies for the rare strains where this was not the case. Frequencies were determined from analysis of at least six colonies; the average and standard deviations are shown. Statistical tests were performed using the Kruskal-Wallis method (Kruskal and Wallis 1952).

Molecular analysis of altered chromosomes.
We analyzed Can R Ade + allelic and mixed colonies for the presence of the specific translocation. Can R Ade + cells were grown in 2-3 mL of rich media, and genomic DNA was prepared and subjected to PCR amplification using ChrVII 403-535 primers (Supplemental Fig. S11 for coordinates). We sequenced the 1.1-kb fragment generated by the translocation to confirm it had the recombination junction shown in Figure 1B (Admire et al. 2006). We typically analyzed four independent cultures by PCR to judge if the translocation was generated. For rad59 mutants that failed to make the translocation, we analyzed >30 mixed colonies. We also used a PCR assay to detect dicentric or acentric chromosomes formed at natural inverted repeats or in the URA module. Again, genomic DNA was isolated from cells grown in rich media, and was subjected to PCR amplification using the appropriate PCR primers (Supplemental Fig. S11). We confirmed that the URA module generates Ura + translocations, using both a PCR assay of the recombination junction (data not shown) and pulse field gels probed with a ChrVII-specific probe (YGL250) (Supplemental Fig. S6). For pulse-field gels, chromosomes were separated (Iadonato and Gnirke 1996) using conditions that optimize for separation of 1100-kb (native) and 1200-kb (translocation) chromosomes.

Real-time PCR quantification
The level of dicentric chromosome was analyzed on a Light-Cycler 1.0 (Roche Diagnostics). A 20-mL amplification mixture contained 2 mL of template DNA, 3 mM MgCl 2 , 5 mM primers (Supplemental Fig. S11), and LightCycler FastStart Master SYBR Green I Mix (Roche Diagnostics). The thermal cycling conditions consisted of 10 min at 95°C, and 40 cycles of 15 sec at 95°C, 10 sec at 58°C, and 90 sec at 72°C. For quantification, standard curves were prepared by serial 15-fold dilutions of PCR-purified ChrVII 403 dicentric DNA fragment. We detected between five and 10 6 dicentric molecules. Crossing points, representing the PCR cycles at which products were detectable, were determined for each sample using the second derivative maximum method (LightCycler 3.5.3 software, Roche Diagnostics). Quality control was ensured by a nontemplate control and determination of the melting point of each PCR product. Only samples in exact concordance with the melting point of a sequence-proofed amplicon were included. In each LightCycler run, the calibrator and unknown sample were measured in duplicate. Variability of the real-time PCRs was 6.3%.


extfieldsofstudy: Biology Medicine

text:
How reliable are G-protein-coupled receptor antibodies?

A cluster of manuscripts in this issue of the Journal highlights a lack of selectivity of 49 antibodies against 19 subtypes of α1- and β-adrenoceptors, muscarinic, dopamine and galanin receptors as well as vanilloid (TRPV1) receptors. Taken together these data demonstrate that lack of selectivity appears to be the rule rather than the exception for antibodies against G-protein-coupled and perhaps also other receptors. Thus, the previously often applied validation of such antibodies by the disappearance of staining in the presence of blocking peptide, i.e. the antigen against which the antibody was raised, alone is insufficient to demonstrate specificity. We propose that receptor antibodies should be validated by at least one of the following techniques: a) disappearance of staining in knock-out animals of the target receptor, b) reduction of staining upon knock-down approaches such as siRNA treatment, c) selectivity of staining in immunoblots or immunocytochemistry for the target receptor vs. related subtypes when expressed in the same cell line and/or d) antibodies raised against multiple distinct epitopes of a receptor yielding very similar staining patterns. Other issues of consideration to obtain reliable results based on receptor antibodies in applications such as immunohistochemistry or immunoblotting are also being discussed.

G-protein-coupled receptors (GPCR) are important regulators of cellular function (Penn 2008). They can be subdivided into classes, families and individual subtypes. Assigning specific functions to a given GPCR often requires determining which subtype is expressed in a specific cell. As GPCR mRNA and protein expression at the cell surface do not necessarily correlate, detection at the protein level is frequently required. GPCR protein detection may also be helpful to identify which cell type within a tissue expresses a given receptor and/or in which subcellular component receptors are found. Moreover, the expression density of GPCR at the cell surface is not a static entity but rather dynamically regulated by a variety of physiological and pathophysiological conditions as well as drug treatment (Insel et al. 2007). A better understanding of such regulation also often requires specific quantification of expression at the protein level. While genetically modified, e.g. HA-or His-tagged receptors, can easily be detected using antibodies against the respective tag (Jongsma et al. 2007), such detection is not applicable to endogenous receptors. Hence, the detection of native GPCR protein typically relies on the use of either specific radioligands, otherwise labelled ligands or antibodies. While radioligands have been extremely useful for studies of many GPCR (Myslivecek et al. 2008;Myslivecek et al. 2007) and continue to emerge, e.g. for nociceptin/orphanin FQ receptors (Ibba et al. 2008), suitable radioligands are missing for many other GPCR, e.g. for β 3 -adrenoceptors (Vrydag and Michel 2007) or free fatty acid receptors (Tanaka et al. 2008). Therefore, antibodies are the only available option to detect many GPCR, and numerous such antibodies are commercially available from various suppliers. If these would be reliable, they would be highly important tools for pharmacological studies. Technical approaches for the generation and validation of receptor antibodies have been reviewed comprehensively (Gupta and Devi 2006;Mackrill 2004).
Doubts about the usefulness of antibodies against GPCR (O'Connell et al. 2006;Pradidarcheep et al. 2008;Rhodes and Trimmer 2006), receptors from the ligand-gated ion channel family (Herber et al. 2004) or transmitter transporters (Chen et al. 2004) have been raised in the past and have even led to the retraction of papers (Hawes and Picciotto 2005). However, these reports have largely been thought to represent isolated cases and did not raise more fundamental questions about the use of receptor antibodies. Hence, receptor antibodies continue to be used widely. In this issue of the Journal, seven groups of investigators report a lack of selectivity of a large number of receptor antibodies for the claimed target receptor. This includes antibodies against subtypes of α 1 -adrenoceptors (Jensen et al. 2009), β-adrenoceptors (Hamdani and van der Velden 2009;Pradidarcheep et al. 2009), dopamine receptors (Bodei et al. 2009), muscarinic receptors (Jositsch et al. 2009;Pradidarcheep et al. 2009), and galanin receptors (Lu and Bartfai 2009). An additional report shows similar problems with antibodies against receptors from the family of ligand-gated ion channels, specifically the TRPV1 receptors (Everaerts et al. 2009), although other antibodies directed against these receptors apparently are selective by hard criteria (Charrua et al. 2009). In total, 49 antisera against 19 distinct receptors have been tested in those studies, 10 of which were tested by more than one group of investigators; in many cases multiple antibodies were investigated per receptor. However, none of them was found to be selective. Taken together, these reports suggest that a lack of selectivity of GPCR and perhaps other receptor antibodies is the rule rather than the exception. In the following we will discuss why so many GPCR antibodies lack selectivity and what we consider to be reliable criteria to demonstrate the specificity of receptor antibodies.
Why do so many GPCR antibodies lack selectivity?
One possible explanation is that subtypes of a given GPCR typically have high degrees of homology and hence antibodies may easily recognize other subtypes within the same family. Indeed, one study reported here shows that antibodies against either β 2 -or β 3 -adrenoceptors recognize rather similar band patterns on immunoblots from cells expressing any of the nine adrenoceptors subtypes (Pradidarcheep et al. 2009). However, the problem apparently goes well beyond the homology of closely related GPCR subtypes. For example, airways express only the M 2 and M 3 subtype of muscarinic receptors (Michel and Parra 2008) but double-knock-out mice lacking both subtypes still stained positive for M 2 and M 3 receptor antibodies (Jositsch et al. 2009). Similarly, triple knock-out mice lacking all three α 1 -adrenoceptor subtypes show similar staining patters as wild-type mice (Jensen et al. 2009). Thus, if lack of selectivity is due to structural similarity between GPCR, this may extend beyond members of a given family to the overall group. If that was true, antibodies raised against less conserved regions of GPCR, e.g. the N-or C-terminal tails may have a better chance for selectivity. However, some findings in this issue of the Journal demonstrate that N-and C-terminal-directed antibodies similarly lack specificity as those directed against more conserved receptor domains (Jensen et al. 2009;Jositsch et al. 2009). Irrespective of such considerations, these problems suggest that many previously published data, including some in this Journal (Otsuka et al. 2008;van Diepen et al. 2008), may require re-interpretation. This shows that irrespective of a good explanation for the lack of selectivity of many GPCR antibodies, it is important to develop better criteria to accept an antibody as specific for its target.

Criteria to demonstrate receptor antibody specificity
The problems with many existing antibodies necessitate revisiting which criteria are acceptable to demonstrate selectivity of an antibody for its claimed cognate receptor. The criterion most often applied in the past is disappearance of staining in the presence of blocking peptide, i.e. the peptide against which the antiserum was raised. However, the examples in this issue of the Journal demonstrate that this is insufficient to demonstrate selectivity as almost all of the tested antibodies had been claimed to be selective based upon blocking peptide testing. A second soft criterion used in the past is that antibodies against different subtypes of a given receptor exhibit distinct staining patters in tissues. However, studies with both knock-out mice (Everaerts et al. 2009;Jensen et al. 2009;Jositsch et al. 2009;Lu and Bartfai 2009;Pradidarcheep et al. 2008) or immunoblots comparing staining to cloned receptor subtypes (Bodei et al. 2009;Hamdani and van der Velden 2009;Pradidarcheep et al. 2009) demonstrate that this also is insufficient to prove selectivity. Rather we propose that at least one of the following four conditions must be met to consider an antiserum to indeed be selective.
Firstly, selectivity of an antibody is likely if its staining disappears in immunohistochemical studies or immunoblots of tissues from animals genetically engineered to lack that receptor. Several studies demonstrate that many available antibodies fail to meet this test (Everaerts et al. 2009;Jensen et al. 2009;Jositsch et al. 2009;Lu and Bartfai 2009;Pradidarcheep et al. 2008). Secondly, animals or cell lines can be treated with genetic tools to knock-down expression of a given receptor, e.g. siRNA; if that leads to a major reduction of staining by a given antibody, this also constitutes reasonable evidence for its specificity. Thirdly, studies with transfection of multiple subtypes of a given receptor into the same host cell line in which the target receptor but not the related subtypes yield positive staining also can provide strong evidence for selectivity (Bodei et al. 2009;Hamdani and van der Velden 2009;Pradidarcheep et al. 2009). Finally, in a more classical immunological approach, it can be considered as reasonable evidence of selectivity if multiple antibodies raised against different epitopes of a GPCR, e.g. N-terminus, intracellular loop and C-terminus, show a very similar staining pattern in immunohistochemistry and/or immunoblotting. Of note, with the possible exception of the third proposed option, all of these criteria may also be applied to antibodies raised against targets other than GPCR. A recent report demonstrates an additional approach based on tagged receptors, in which a GPCR antibody detects the same protein as the antibody against the tag (Miyauchi et al. 2009); however, this approach needs to be validated in further studies using the hard criteria described above.

Additional considerations for the validation of receptor antibodies
The apparent selectivity or lack thereof for a receptor antibody may also depend on other factors. For example, in immunohistochemical studies the recognition pattern of an antiserum may depend on the protocols used for tissue fixation (Jensen et al. 2009). Also, an antibody raised against a recombinant protein or part of it might be highly specific in immunohistochemistry, but is not able to detect the protein after its threedimensional structure has been destroyed, e.g. by solubilization in Laemmli buffer.
Selectivity data based upon immunoblots may be liable to additional problems when used for other purposes. Thus, even if a band of appropriate size is present only in extracts of cells expressing the target receptor but not in those expressing related subtypes, this does not necessarily demonstrate the usefulness of such antibodies for immunohistochemistry. As immunohistochemistry relies on the idea that all or at least most staining comes from the target protein, staining of additional bands in immunoblots may be a significant source of error when using the same antiserum in immunohistochemistry. Thus, the number of detected bands on an immunoblot may to a large degree depend protein loading of the gel, and only antibodies exhibiting immunoblot specificity over a wide range of loading densities may have sufficient specificity for immunohistochemistry. A variation of this idea is that an antibody may exhibit "specificity" when tested on cell lines with marked overexpression but not when much lower physiological expression densities are assessed, i.e. has poor sensitivity for its target receptor. This is particularly true if evidence for specificity is based solely on the presence of a band on immunoblots from overexpressing as compared to mock-transfected cells.

Conclusions and future editorial policy
As a lack of selectivity appears to be the rule rather than the exception with GPCR antibodies, the scientific community should consider which requirements are agreed upon to consider an antibody to have validated selectivity for its claimed cognate receptor. To initiate such discussion, we propose four criteria, of which at least one should be fulfilled as reasonable evidence for selectivity. At least for this Journal, this will become an editorial policy for all manuscripts which use GPCR antibodies. We additionally propose that similarly rigorous validation approaches should also be applied to antibodies against targets other than GPCR. Finally, the various reports in this issue of the journal imply that lack of selectivity of receptor antibodies is not a problem of one or two specific commercial suppliers but rather appears to be applicable to all antibody sources, both commercial and academic. As the data in this issue of the journal raise more questions than provide answers, the journal welcomes scientific comments from the academic community and commercial antibody suppliers with regard to strategies for improved antibody selectivity and/or criteria to demonstrate such selectivity.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


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Ubiquitous RNA-dependent RNA polymerase and gene silencing

The discovery of a ubiquitous RNA-dependent RNA polymerase raises new questions about small RNA silencing mechanisms.

The phenomenon of the silencing of transgenes together with homologous genes in the host genome was first discovered some two decades ago in plants [1][2][3]. This silencing was eventually found to involve either posttranscriptional destruction of the transgene mRNAs [2,3] or prevention of their transcription [1]. The biological rationale for these processes was proposed to be a defense against viruses and transposable elements. Doublestranded RNA (dsRNA) is a target of the silencing mechanism and many viruses produce such RNA as part of their life cycle. Similarly, transposable elements produce aberrant RNAs that enter the silencing pathway and this prevents transposition. This host defense concept has since been supported by a wide variety of evidence. The inde pendent discovery in Caenorhabditis elegans that introduced 'sense' RNAs could cause gene silencing led to the development of the technique of RNA interference (RNAi) [4]. The finding that the active ingredient in triggering RNAi was a dsRNA ignited a revolution in genetics that continues to this day [5].
An interesting aspect of post-transcriptional silencing in plants and worms is that silencing can spread throughout the organism from the site of introduction of the dsRNA. This spread depends on the activity of an RNA-dependent RNA polymerase (RdRP), which amplifies dsRNA to produce more copies, which are then cleaved by the enzyme Dicer to produce small interfering RNAs (siRNAs) [6] that target mRNAs of similar sequence for destruction. The genomes of Drosophila and mammals show no evidence of such an RdRP, and the spread of RNAi from one tissue to another is indeed limited in these animals. Nevertheless, other aspects of post-transcriptional silencing suggested that some type of RdRP activity might be critical in flies and humans as well. For example, aberrant single-stranded RNAs (ssRNAs) are also subject to silencing and so presumably must be converted to a doublestranded form [7,8]. Indeed, an RdRP activity was reported in extracts of Drosophila some years ago [9], but the conventional wisdom based on the genomic data held that such an activity did not exist in flies and mammals.
In a recent report, Lipardi and Paterson [10] now define a gene in Drosophila whose product has a newly recognized RNA-dependent RNA polymerase activity, which provides a potential solution to the outstanding issues in posttranscriptional silencing noted above. Moreover, the authors show that this gene product is a multifunctional protein, which in one of its roles is a member of the transcriptional RNA polymerase II elongator complex, and that, unlike the canonical RdRP, it is present in all eukaryotes examined so far. Its ubiquity raises many interesting questions about other possible roles for silencing by small RNAs other than as a defense against viruses and transposons.

Elp1 is a novel RNA-dependent RNA polymerase
Lipardi and Paterson [10] sought to purify the previously reported RdRP activity [9] from Drosophila embryo extracts. They then tested the candidate proteins identified from a partial purification to determine whether they could inhibit RNAi of green fluorescent protein (GFP) in cultured Drosophila cells engineered for that purpose. They found that knockdown of the Drosophila homolog of the RNA polymerase II core elongator complex subunit Elp1 in the cultured cells inhibited RNAi. Knockdown of other members of the elongator complex had no effect on GFP silencing, and ectopically expressed Elp1 protein was clearly visualized in the cytoplasm. The authors produced recombinant Elp1 protein and showed that it could generate dsRNA using ssRNA as a template in both primerindependent and primer-dependent reactions in vitro. They then coexpressed different combinations of Elp1, Argonaute-2 (which binds siRNAs), Dicer-2 (which cleaves dsRNA to siRNAs) and Dicer-1 (which cleaves microRNA precursors to the mature form) in cultured Drosophila cells, and looked for protein-protein interactions. The strongest interaction was between Elp1 and Dicer-2, which demonstrates a connection to RNAi given the central role of Dicer-2 in producing siRNAs. The levels of certain microRNAs were assayed in Elp1-depleted cells and found not to be affected, indicating a specificity of Elp1 for the RNAi pathway. Knockdowns of Elp1 by RNAi [10] produced an increase in the expression of a set of retrotransposons, which were similarly affected by knockdown of Dicer-2. A corres ponding reduction in the levels of siRNAs for these trans posons was also found. RNAi knockdown of Elp1 specifically reduced the levels of antisense RNAs for the affected transposons, whereas depletion of Dicer-2 did not, thus strengthen ing the argument that Elp1 provides an RNAdependent RNA polymerase activity that generates the antisense strand. Unlike the gene for canonical RdRP, the elp1 gene is conserved in all eukaryotes examined [10]. Recombinant Elp1 produced from fission yeast, C. elegans and human sequences was shown to have RdRP activity in vitro. Thus, this activity is present in species with and without the canonical RdRP gene.
The elucidation of an RdRP activity that can act without a primer helps to clarify findings that 'aberrant' ssRNAs can be degraded by the silencing process [7,8,11]. This posttranscriptional silencing in the absence of an antisense copy or a foldback structure that could produce a dsRNA to serve as a substrate for Dicer had previously posed a conun drum. The discovery of Elp1 RdRP activity provides a possible solution, given that it could potentially convert the aberrant ssRNAs to a double-stranded structure without an antisense primer, but such a scenario needs to be tested experimentally.

Questions for the future
Small RNAs are also involved in transcriptional silencing [7,11,12], and there is increasing evidence that interaction of RNA polymerase II (PolII) with the small RNA machinery is intimately involved in this silencing. The first indication came from fission yeast (Schizosaccharomyces pombe) in which mutation in the largest and the second largest of the polymerase subunits impaired transcriptional silencing [13,14]. In plants, novel RNA polymerases [15] have been found that are dedicated to small-RNA-based transcriptional silencing. The finding of Lipardi and Paterson [10] of a protein with RdRP activity associated with the PolII core elongator complex raises the question of whether this protein is involved in transcriptional silencing. It may indeed play no such role in the polymerase complex, given that it has a cytoplasmic localization when involved in post-transcriptional silencing. However, the evidence of a role for PolII in silencing suggests that investigating the possibility would be an important future direction for work on gene silencing.
Somewhat ironically, the Elp1 protein has a much broader phylogenetic distribution among eukaryotes than the so-called 'canonical' RdRP [10]. So another important ques tion raised by these findings is whether the Elp1 RdRP activity is present in all eukaryotes, including those with the canonical RdRP, and what is the relationship of the two activities in gene-silencing mechanisms? The fact that mutations in the canonical gene produce a recognizable effect on silencing is a classic genetic argument that the two functions will not be completely redundant. Nevertheless, this deserves more investigation.
In addition to the siRNA products of Dicer, Drosophila and mammals produce other short RNAs called piRNAs that associate with the PIWI family of Argonaute proteins [16][17][18]. piRNAs are found in abundance in germline cells but have also been reported in somatic tissues. One proposed role of these small RNAs is as a defense against transposable elements. The terminal structure of piRNAs is not like that produced by Dicer [19] and so they are likely to be produced by a Dicer-independent mechanism. Different members of the PIWI protein family show a preference for binding to either the sense or antisense versions of retrotransposon piRNAs [17,18]. The complemen tary sequence arrangements of piRNAs had suggested that the sense and antisense versions serve as guides for different Argonaute proteins to cleave them to their characteristic 25-bp length. The presumed absence of an RdRP activity in flies and mammals figured in the development of this idea because there was no known mechanism by which dsRNA intermediates could be generated. With the revelation that such an activity exists, an interesting direction of investigation would be to test whether production of piRNAs does in fact involve a dsRNA intermediate. Indeed, given that piRNAs are implicated in transcriptional silencing [20,21], interconnections among PolII, Elp1 and the PIWI family of Argonaute proteins in silencing processes would be worth exploring.
Finally, the finding of a protein with RdRP activity that associates with PolII and is found in all eukaryotes might suggest an expansion of the raison d'être for small RNAmediated silencing mechanisms. It might well be that small RNAs have functions in RNA proofreading and quality control that act to silence the transcription of DNA sequences encoding unwanted or aberrant RNAs. This mechanism would silence transposable elements as just one aspect of its broader role in gene expression. Nevertheless, the antiviral functions of small RNAs act post-transcriptionally and the relationship of these to transcriptional silencing is still in need of clarification. These potential interconnections raise new questions for the ever-enlarging field of small RNAs.