Allosteric Regulation of RecA Protein Function Is Mediated by Gln194 *

Binding of ATP to the RecA protein induces a high affinity DNA binding required for activation of enzyme function. Screens for in vivo recombination and repressor cleavage activities show Gln194 to be intolerant of all substitutions. Analyses of three mutant proteins (Q194N, Q194E, and Q194A) show that although basal enzyme function is maintained, each protein no longer displays an ATP-induced increase in DNA binding affinity. High salt activation of RecA function is also disrupted by these mutations. In contrast, ATP-induced changes in the oligomeric structure of RecA are maintained in the mutant proteins. These results demonstrate that Gln194 is a critical “allosteric switch” for ATP-induced activation of RecA function but is not the exclusive mediator of ATP-induced changes in RecA.

The Escherichia coli RecA protein is a multifunctional enzyme that plays a central role in the processes of recombinational DNA repair, homologous genetic recombination, and the cellular SOS response to DNA damage (1)(2)(3). Each of these activities exhibits a common initial or activating step, formation of a RecA-ATP-ssDNA 1 nucleoprotein filament (4 -6). The binding of RecA to ssDNA is regulated in a classic allosteric fashion, whereby the binding of ATP induces a high affinity DNA binding state of the protein (7,8). In the presence of ADP, or in the absence of cofactor, RecA exhibits a low affinity (Ͼ20 M) for ssDNA (7).
The crystal structure of the helical RecA protein filament has been solved in both the absence and presence of ADP (9, 10) and displays a helical pitch (82.7 Å) which is intermediate between the inactive nucleotide-free form (Ϸ70 Å) and the active ATPbound form (Ϸ95 Å) as determined by electron microscopy (reviewed in Ref. 11). Despite this, the ATP binding site shows a remarkable conservation of structure compared with several other nucleotide-binding proteins (e.g. p21 ras , EF-Tu, and adenylate kinase), and Story and Steitz (10) were able to model specific determinants of both ATP binding and hydrolysis in the RecA structure. In addition, the structure provided valuable insight into a possible allosteric mechanism for ATP-induced high affinity binding to ssDNA. The carboxyamide side chain of Gln 194 extends into the ATP binding site and would be in very close proximity to the ␥-phosphate of bound ATP (Fig.  1). Gln 194 immediately precedes one of two disordered loops in the structure (L2, residues 195-209) that were proposed to be part of the DNA binding sites (9). Recent work has, in fact, provided strong evidence that L2 comprises all or a large part of the ssDNA binding site within RecA (12)(13)(14). Story and Steitz (10) proposed that upon ATP binding Gln 194 interacts with the nucleotide ␥-phosphate thereby causing L2 to assume a conformation with high affinity for ssDNA. Upon ATP hydrolysis this interaction would be lost, returning L2 to a low affinity DNA binding conformation.
In this study, we show that mutations at Gln 194 prohibit the formation of a high affinity ssDNA binding state when ATP is bound. In addition, mutations at Gln 194 block the high salt activation of RecA function. These results indicate that Gln 194 is an important "on-off" switch required for the general activation of RecA function.

EXPERIMENTAL PROCEDURES
Materials-Labeled NTPs and dNTPs were from NEN Life Science Products. PEI-cellulose chromatography plates were from J. T. Baker Inc. Single-stranded RV-1 DNA, an M13 derivative, was used for in vitro ATPase and repressor cleavage assays and was purified as described (15). An 86-base oligonucleotide used in the ssDNA binding assays and mutagenic oligonucleotides were made with an Applied Biosystems 392 DNA/RNA synthesizer. Double-stranded DNA (dsDNA) used in both the ATPase and dsDNA binding assays was NcoI-linearized pTRecA220 (Ϸ5, 500 base pairs (16)). LexA protein was a generous gift from Dr. John Little and Donald Shepley (Dept. of Biochemistry, University of Arizona). Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase, and Klenow DNA polymerase I large fragment were from New England Biolabs. Sequenase version 2.0 was from U. S. Biochemical Corp. Nitrocellulose filters were from Schleicher and Schuell. Isopropyl-1-thio-␤-D-galactopyranoside and mitomycin C were from Sigma.
Mutagenesis-Mutations were introduced at position 194 using a modification of a previously described cassette mutagenesis procedure (17). Two 81-base oligonucleotides corresponding to the top and bottom strands encoding residues Thr 186 to Asn 213 were synthesized such that codon 194 read NN(G/C) and all other bases were the wild type recA sequence. Oligonucleotides were annealed, and the resulting cassette was ligated into AflIII/MluI-digested pTRecA332, a derivative of pTRecA322 (18) containing a unique AflIII site at position 187 and a unique MluI site at position 214. Plasmids were transformed into a ⌬recA strain, DE1663Ј (18), and colonies were selected on LB-ampicillin plates. Amino acid substitutions were determined by DNA sequence analysis.
Recombinational DNA Repair Activity in Vivo-The recombinational DNA repair activity of each mutant protein was determined using two genetic screens as described previously (18), cell survival in the presence of mitomycin C and cell survival following exposure to different doses of UV light.
Determination of the Size of Mutant RecA Protein Oligomers-The oligomeric distribution of wild type and mutant RecA proteins was determined using gel filtration chromatography as described previously (19).
Purification of Wild Type and Mutant RecA Proteins-Wild type and mutant proteins (Q194A, Q194E, and Q194N) were purified using a previously described method (20) and were determined to be Ͼ95% pure * This work was supported by National Institutes of Health Grant GM44772 (to K. L. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
as judged by Coomassie-stained gels. Proteins were quantitated using the Bio-Rad protein assay kit and by optical density, using an extinction coefficient of ⑀ 280 ϭ 0.59 mg Ϫ1 ⅐ml (21). No exonuclease activity was detected in any of these preparations, even at elevated concentrations of RecA (50 M) in the nuclease assay. None of these mutations has any deleterious effect on the overall folded structure or thermal stability of the protein. Wild type RecA and each mutant protein showed identical circular dichroism profiles and measurements of 222 nm as a function of temperature (5-90°C) gave a T m ϭ 53 Ϯ 1°C for all 4 proteins (data not shown).
RecA-mediated LexA Cleavage-RecA-mediated cleavage of the LexA repressor was measured in vivo using strain DE1663Ј as described previously (18). The ability of purified wild type and mutant RecA proteins to mediate the autocleavage of the LexA repressor in vitro was assessed in the presence of ssDNA as described previously (20) and in the presence of high salt using the following modifications of a previously described method (22). Reaction buffer contained 50 mM K 2 HPO 4 / KH 2 PO 4 , 10 mM Mg(OAc) 2 , 0.6 M NaOAc, 5 mM ATP␥S, 5% glycerol and was brought to a final pH of 7.0. Reactions (60 l) were performed at 37°C and contained RecA protein (0.5 mg/ml) which had been preincubated with 5 mM ATP␥S. LexA protein was added to a final concentration of 1.0 mg/ml. During the time course samples (8 l) were removed and EDTA was added to 100 mM. Intact LexA and cleavage products were resolved on a 15% SDS-polyacrylamide gel, stained with Coomassie Brilliant Blue, and quantitated using scanning densitometry (20).
ATPase Activity-Hydrolysis of ATP was measured under 3 different conditions, in the presence of ssDNA, dsDNA, or 1.8 M NaCl. The ssDNA-dependent ATPase activity was measured as described previously (20,23). The dsDNA-dependent ATPase activity was measured essentially as described (23). Reaction mixtures contained 20 mM sodium maleate (pH 6.2), 10 mM ssDNA Binding-Nitrocellulose filter binding assays were performed using a variation of a described procedure (25) and an apparatus similar to that described by Wong and Lohman (26). Filters were prepared by soaking in 0.4 M KOH for 10 min followed by several washes with double distilled H 2 0 until the pH approximated 7.0. Filters were equilibrated in binding buffer for at least 1 h prior to use. Reaction mixtures (50 l) contained binding buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM DTT, 0.5 mM EDTA, 20 mM NaCl), 40 M 5Ј-end-labeled ssDNA (concentration of bases), and where indicated, 0.5 mM ATP␥S. Reactions were started with the addition of the indicated amounts of protein and incubated at 37°C for 15 min. Samples were applied to the filter under suction, and the filters were washed with 2.0 ml of binding buffer containing 150 mM NaCl. Filters were air-dried, and bound DNA was quantitated by analyzing air-dried filters with a Molecular Dynamics PhosphorImager and ImageQuant software (v5.6).
dsDNA Binding-Nitrocellulose filter binding assays were performed using a variation of a previously described procedure (25). Mixtures were placed on ice, 4 cm from an 8-watt mercury vapor lamp (Sylvania G8T5), and irradiated for 45 min. Crosslinked products were resolved on a 10% SDS-polyacrylamide gel and analyzed using a Molecular Dynamics PhosphorImager and ImageQuant software (v5.6).

RESULTS AND DISCUSSION
RecA Activity in Vivo-We obtained 16 of 19 possible substitutions at Gln 194 , including a conservative Asn, an isosteric Glu, and an Ala. 2 In vivo screens for recombinational DNA repair and LexA cleavage showed that these activities are completely dependent on the wild type Gln residue (data not shown). Each of the 16 mutants showed no survival greater than the ⌬recA control in the presence of mitomycin C or following exposure to UV, even under low dose conditions (0.3 g/ml mitomycin C or 0.67 J/m 2 /s UV light). In addition, the LexA coprotease activity of each mutant was completely inhibited, a result which was confirmed by in vitro LexA cleavage assays (see below).
ssDNA Binding-RecA exhibits two distinct binding affinities for ssDNA, a low affinity in the absence of any nucleotide cofactor or the presence of ADP, and a high affinity in the presence of cofactor (7,8). We show that in the absence of nucleotide wild type RecA protein binds to ssDNA with an apparent K d Ϸ20 M (Fig. 2A). Each mutant protein tested, Q194A, Q194E, and Q194N, exhibits a comparable apparent affinity, although the Q194N protein appears to have a slightly reduced binding capacity ( Fig. 2A). In the presence of ATP␥S, however, a dramatic increase is seen in the affinity of wild type RecA for ssDNA compared with the mutant proteins (Fig. 2B). Wild type RecA now binds the ssDNA substrate with an apparent K d Ϸ1 M, whereas none of the 3 mutant proteins shows any change in affinity for ssDNA. These results indicate that the mutations at position 194 render the proteins unable to undergo the ATP-induced transition required for high affinity ssDNA binding.
ATP Binding-An essential control was to test the ability of the mutant proteins to bind ATP. Using a UV crosslinking procedure we show that ATP binding by each of the 3 mutant proteins (Q194A, Q194E, and Q194N) is very similar to that of wild type RecA (Fig. 3). Proteins that had been heat-denatured prior to UV exposure show no crosslinking. These results show that the inability of the mutant proteins to assume a high affinity ssDNA binding state is not due simply to a defect in ATP binding.
ATP-induced Effects on the Oligomeric Distribution of RecA-In solution RecA protein exists as a heterogeneous population of oligomers, ranging in size from monomers, dimers, and hexamer-sized rings to larger filaments and bundles of filaments (27)(28)(29)(30). In the presence of ATP this distribution is shifted such that bundles of filaments are disrupted and the average filament length is shorter (27)(28)(29). We used a recently developed gel filtration assay (19) to assess any effect that mutations at position 194 may have on the oligomeric properties of RecA, as well as the effect of ATP on the oligomeric distribution. Our results show that substitution of Gln 194 with Ala, Glu, or Asn has no effect on the oligomeric distribution of RecA and importantly, that each mutant protein shows an ATP-induced shift in this distribution identical to that observed for wild type RecA (Fig. 4). Addition of ATP␥S shifts the peak representing long RecA filaments and bundles to a smaller size (Fig. 4 and Ref. 19). These results again show that each mutant RecA protein is capable of binding ATP and that conformational changes associated with the ATP-induced shift in the distribution of RecA oligomers are maintained and therefore not transmitted through Gln 194 .
ATPase Activity-The ATPase activity of RecA protein is stimulated upon binding to either ssDNA or dsDNA (23). In the absence of DNA wild type RecA exhibits a low level ATPase activity at pH 7.5 (V i /E ϭ 0.2) whereas in the presence of a ssDNA cofactor activity increases significantly (V i /E ϭ 17.4; Fig. 5). While the basal rate of ATP turnover for the mutant proteins is similar to that of wild type RecA (V i /E Ϸ0.2; Fig.   5A), no ssDNA-dependent increase in activity is observed (Fig. 5B).
At pH 6.2 the catalytic efficiency of RecA increases approximately 30-fold in the presence of dsDNA (23). Each of the Gln 194 mutant proteins maintains a wild-type level of DNAindependent ATPase activity at pH 6.2 (V i /E ϭ 0.5). However, an increase in the presence of dsDNA was seen only with wild type RecA (data not shown). Control experiments showed that wild type and mutant proteins bound equivalent low levels of dsDNA in the absence of ATP␥S, yet only wild type RecA displayed a significant ATP-dependent increase in dsDNA binding (data not shown).
These results demonstrate that mutations at Gln 194 block the DNA-dependent activation of ATPase activity but do not affect the basal level of DNA-independent ATP turnover.
LexA Coprotease Activity-In the presence of both nucleotide and ssDNA cofactors RecA mediates the autoproteolysis of the LexA repressor (5). While wild type RecA catalyzes a significant level of LexA autoproteolysis (95% cleaved/40 min) this activity is completely lacking in each of the 3 mutant proteins (data not shown).
High Salt Activation of RecA Activities-In the absence of any DNA cofactor, but in presence of high salt, wild type RecA protein is activated for both ATP hydrolysis (24) and LexA cleavage (22). Because mutations at Gln 194 prohibit high affinity DNA binding required for both of these activities we determined whether the Q194A, Q194E, and Q194N mutations also affected high salt activation of RecA function. We found that although the ATPase activity of wild type RecA was greatly stimulated in the presence of 1.8 M NaCl (V i /E ϭ 23) the 3 mutant proteins showed only very low activity under these conditions (Fig. 6). Similarly, in the presence of 0.6 M NaOAc wild type RecA catalyzed a significant level of LexA autoproteolysis (50% cleaved/40 min), yet this activity was lacking in each mutant protein (data not shown).
These results demonstrate that the wild type Gln 194 side chain is necessary for the general activation of RecA function and supports the suggestion by Pugh and Cox (24) that salt activation of RecA is "functionally mimicking the ionic interaction of the protein with DNA." In the presence of ATP and elevated salt concentrations, if 3 to 4 anions bind to the same sites within RecA as do phosphate groups on the DNA backbone (24,31), our data show that Gln 194 mediates the ATPinduced occupancy of these sites giving rise to an activated RecA-ATP-DNA (or RecA-ATP-salt) complex. In future studies it would be interesting to determine the ion occupancy of the Gln 194 mutant proteins. Our data identify Gln 194 as a NTP-binding site "␥-phosphate sensor" in that mutations at this residue disrupt only the ATP-induced increase in RecA activities and not the basal level functions or properties of the protein. For example, the low level DNA-independent ATP hydrolysis seen with wild type RecA is maintained for each of the Gln 194 mutant proteins indicating that Gln 194 is not an important component of the intrinsic RecA ATPase catalytic mechanism. In addition, Gln 194 appears not to be an important determinant of ATP binding because Asn, Glu, and Ala mutations show binding profiles similar to wild type RecA. We also show that each of the 3 mutant proteins maintains a low affinity binding to ssDNA in the absence of ATP similar to wild type RecA, and therefore, Gln 194 is not a determinant of this DNA binding property. We cannot necessarily exclude the possibility that Gln 194 interacts with DNA in the activated RecA-ATP-DNA complex. Using synthetic peptides corresponding to residues 193-212, which include the entire L2 region, Voloshin et al. (14) identified position 203 as an important determinant of DNA binding. Studies using peptides with substitutions at Gln 194 could assist in defining a potential role for this residue in DNA binding.
Although our data demonstrate the importance of Gln 194 in transmitting allosteric information within the RecA protein structure it certainly does not exclude other residues, perhaps within the L2 DNA binding region, as also participating in ATP-induced allosteric effects. We note that specific ATP-induced changes in the oligomeric structure of RecA are not blocked by mutations at Gln 194 (Fig. 4). Therefore, other residues must play important roles in mediating nucleotide-induced changes in RecA structure and/or function as the protein progresses through the catalytic cycle.