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• W2004642972 abstract "Essential to the two distinct cellular events of genetic recombination and SOS induction in Escherichia coli, RecA protein promotes the homologous pairing and exchange of DNA strands and the proteolytic cleavage of the LexA repressor, respectively. Since both of these activities require single-stranded DNA (ssDNA) and ATP, the inter-relationship between these reactions was investigated and found to display many parallels. The extent of active complex formed between RecA protein and M13 ssDNA, as measured by both ATP hydrolysis and LexA proteolysis, is stimulated in a similar manner by either a reduction in magnesium ion concentration or the presence of single-stranded DNA binding (SSB) protein. However, unexpectedly, SSB protein inhibits both LexA proteolysis and ATP hydrolysis (in assays containing repressor) at concentrations of RecA protein that are substoichiometric to the ssDNA, arguing that LexA repressor affects the competition between RecA and SSB proteins for limited ssDNA binding sites. Additionally, attenuation of LexA repressor cleavage in the presence of double-stranded DNA or by an excess of ssDNA suggests that interaction of the RecA nucleoprotein filament with either LexA repressor or a secondary DNA molecule is mutually exclusive. The significance of these results is discussed in the context of both the regulation of inducible responses to DNA damage, and the competitive relationship between the processes of SOS induction and genetic recombination. Essential to the two distinct cellular events of genetic recombination and SOS induction in Escherichia coli, RecA protein promotes the homologous pairing and exchange of DNA strands and the proteolytic cleavage of the LexA repressor, respectively. Since both of these activities require single-stranded DNA (ssDNA) and ATP, the inter-relationship between these reactions was investigated and found to display many parallels. The extent of active complex formed between RecA protein and M13 ssDNA, as measured by both ATP hydrolysis and LexA proteolysis, is stimulated in a similar manner by either a reduction in magnesium ion concentration or the presence of single-stranded DNA binding (SSB) protein. However, unexpectedly, SSB protein inhibits both LexA proteolysis and ATP hydrolysis (in assays containing repressor) at concentrations of RecA protein that are substoichiometric to the ssDNA, arguing that LexA repressor affects the competition between RecA and SSB proteins for limited ssDNA binding sites. Additionally, attenuation of LexA repressor cleavage in the presence of double-stranded DNA or by an excess of ssDNA suggests that interaction of the RecA nucleoprotein filament with either LexA repressor or a secondary DNA molecule is mutually exclusive. The significance of these results is discussed in the context of both the regulation of inducible responses to DNA damage, and the competitive relationship between the processes of SOS induction and genetic recombination. Insults inflicted upon DNA represent a serious challenge to cellular survival in all living organisms. The RecA protein has several distinct roles in maintaining genetic integrity within Escherichia coli. In promoting the recognition and subsequent exchange of strands between homologous DNA molecules (for a recent comprehensive review see 1Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Google Scholar and references therein), the RecA protein is central to recombinational repair events. In addition, the RecA protein coordinates the cellular response to factors that confer DNA damage or interfere with DNA replication by inducing the expression of a set of unlinked genes, recA included, comprising the SOS regulatory system (2Little J.W. Mount D.W. Cell. 1982; 29: 11-22Google Scholar, 3Walker G.C. Annu. Rev. Biochem. 1985; 54: 425-457Google Scholar, 4Witkin E.M. Biochimie (Paris). 1991; 73: 133-141Google Scholar). Initiation of the SOS response occurs when RecA protein becomes activated to stimulate cleavage of the LexA protein, the transcriptional repressor of SOS regulon genes (5Witkin E.M. Bacteriol. Rev. 1976; 40: 869-907Google Scholar). The LexA repressor is not the only target of activated RecA protein; cleavage of lytic repressors from various lambdoid bacteriophage accounts for prophage induction (6Roberts J.W. Roberts C.W. Craig N.L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4714-4718Google Scholar), while maturation of the mutagenesis factor (7Shinagawa H. Iwasaki H. Kato T. Nakata A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1806-1810Google Scholar), UmuD protein, is necessary for bypass of lesions during DNA replication (8Rajagopalan M. Lu C. Woodgate R. O'Donnell M. Goodman M.F. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10777-10781Google Scholar). Repair of DNA damage and alleviation of blocks to replication serve to eliminate the signal that activates the RecA protein; consequently, as intact LexA repressor reaccumulates, the expression of SOS genes return to normal uninduced levels. The ability to interact with DNA and a nucleotide triphosphate cofactor is fundamental to the remarkably diverse biochemical activities of the RecA protein. In a series of kinetically discernible steps, in vitro, the RecA protein promotes homologous pairing and transfer of strands between a variety of DNA substrates, provided one possesses some single-stranded character (1Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Google Scholar, 9Kowalczykowski S.C. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 539-575Google Scholar). The elementary stage of the reaction involves assembly of a nucleoprotein complex, through the nonspecific and cooperative binding of the RecA protein to a fully or partially ssDNA 1The abbreviations used are: ssDNAsingle-stranded DNAdsDNAdouble-stranded DNAATPγSadenosine 5′-[thio] triphosphateSSBsingle-stranded DNA bindingpoly(dT)polydeoxy(thymidylic acid)etheno M13 ssDNAmodified M13 ssDNA containing 1,N6-ethenoadenosine and 3,N4-ethenocytidine residuesIODintegrated optical density. molecule, in the presence of ATP, that is capable of searching for and establishing homologous contacts with duplex DNA. Similarly, initial studies examining proteolysis of phage repressors by RecA protein also revealed a requirement for both nucleotide triphosphate and single-stranded polynucleotide (6Roberts J.W. Roberts C.W. Craig N.L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4714-4718Google Scholar, 10Craig N.L. Roberts J.W. Nature. 1980; 283: 26-29Google Scholar, 11Phizicky E.M. Roberts J.W. J. Mol. Biol. 1980; 139: 319-328Google Scholar). Cleavage of the LexA (12Little J.W. Edmiston S.H. Pacelli L.Z. Mount D.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3225-3229Google Scholar) and UmuD (13Burckhardt S.E. Woodgate R. Scheuermann R.H. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1811-1815Google Scholar) proteins also occurs, in vitro, when RecA protein binds to ssDNA and either ATP, dATP, or the relatively non-hydrolyzable analogue, ATPγS. However, in contrast to a traditional protease, the RecA nucleoprotein filament plays an indirect role as a “coprotease” in stimulating the specific autodigestion of these target proteins (14Little J.W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1375-1379Google Scholar, 15Little J.W. Biochimie (Paris). 1991; 73: 411-421Google Scholar). The relative abundance of free RecA protein and ATP in uninduced cells implicates ssDNA as the critical component responsible for in vivo activation of the RecA protein. Consistent with this proposal, the production of single-stranded regions in damaged DNA through either the helicase activity of the RecBCD protein (16Chaudhury A.M. Smith G.R. Mol. Gen. Genet. 1985; 201: 525-528Google Scholar) or ongoing DNA replication (17Sassanfar M. Roberts J.W. J. Mol. Biol. 1990; 212: 79-96Google Scholar) is a prerequisite for derepression of the SOS regulon by various inducing treatments. The role of ssDNA as the activating signal, in vivo, is confirmed by observations that infection by mutant filamentous phage that are defective in complementary (minus) DNA strand synthesis induces the SOS response (18Higashitani N. Higashitani A. Roth A. Horiuchi K. J. Bacteriol. 1992; 174: 1612-1618Google Scholar). single-stranded DNA double-stranded DNA adenosine 5′-[thio] triphosphate single-stranded DNA binding polydeoxy(thymidylic acid) modified M13 ssDNA containing 1,N6-ethenoadenosine and 3,N4-ethenocytidine residues integrated optical density. The binding of RecA protein to ssDNA and ATP yields a ternary complex that is the functional species in both the homologous pairing of DNA and the proteolytic cleavage of the LexA repressor; since a parallel consequence of this binding is the hydrolysis of ATP, the formation and properties of the ternary complex can be monitored indirectly by measuring the ssDNA-dependent ATP hydrolysis activity of the RecA protein. To further our understanding of the mechanisms of and relationship between the cellular processes of SOS induction and genetic recombination, we have compared cleavage of the LexA repressor by this ternary complex to hydrolysis of ATP. As anticipated, considering the shared requirement for ssDNA and ATP, the LexA repressor cleavage and ATP hydrolysis activities of RecA protein display many parallels. The ability of SSB protein to stimulate maximal rates of RecA protein-promoted LexA proteolysis and ATP hydrolysis, in vitro, is consistent with it being genetically critical in both the response to and the recombinational repair of DNA damage (1Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Google Scholar, 19Meyer R.R. Laine P.S. Microbiol. Rev. 1990; 54: 342-380Google Scholar). Unexpectedly, however, demonstration that the LexA repressor influences the competition between RecA and SSB proteins for limited ssDNA binding sites identifies an additional manner by which inducible responses to DNA damage may be regulated. Despite many earlier studies focusing on either homologous DNA pairing or coprotease activities, it remained unclear whether proteolytic cleavage is independent of or competitive with other RecA protein-promoted reactions. Our characterization of the inhibition of LexA proteolysis by dsDNA and excess ssDNA provides direct biochemical evidence that supports the idea, based on electron microscopy (20Yu X. Egelman E.H. J. Mol. Biol. 1993; 231: 29-40Google Scholar), that interactions between the RecA nucleoprotein filament and either the LexA repressor or a secondary DNA molecule are mutually exclusive. Thus, the cellular processes of SOS induction and genetic recombination may be intrinsically competitive in nature. Studies using a non-cleavable mutant of the LexA repressor protein, presented in the accompanying paper (21Harmon F.G. Rehrauer W.M. Kowalczykowski S.C. J. Biol. Chem. 1996; 271: 23874-23883Google Scholar), reinforce these views. All chemicals were reagent grade; solutions were made using Barnstead NANOpure water. ATP, dATP, and ATPγS were purchased from Pharmacia Biotech, Inc., Sigma, and Boehringer Mannheim, respectively. The nucleotides were dissolved as concentrated stocks at pH 7.5, and their concentrations were determined spectrophotometrically using an extinction coefficient of 1.54 × 104M−1 cm−1 at 260 nm. The RecA protein was purified from E. coli strain JC12772 using a modified preparative procedure 2S. C. Kowalczykowski, manuscript in preparation. based on spermidine acetate precipitation (22Griffith J. Shores C.G. Biochemistry. 1985; 24: 158-162Google Scholar). SSB protein was purified from E. coli strain RLM727 as described (23LeBowitz J. Biochemical Mechanism of Strand Initiation in Bacteriophage Lambda DNA Replication. Johns Hopkins University, Baltimore1985Google Scholar). Protein concentrations were determined using molar extinction coefficients of 2.7 × 104M−1 cm−1 for RecA protein and 3.0 × 104M−1 cm−1 for SSB protein, both at 280 nm. LexA repressor was purified from strain JL652 using essentially the protocol of Schnarr et al. (24Schnarr M. Pouyet J. Granger-Schnarr M. Daune M. Biochemistry. 1985; 24: 2812-2818Google Scholar) with the following modifications; phosphocellulose fractions containing LexA repressor were assayed to detect, and pooled to avoid, an overlapping elution of deoxyribonuclease activity; following dialysis, a step elution from a Q-Sepharose column using 100 mM NaCl resolved the intact LexA repressor (≥98% pure based on SDS-polyacrylamide gel electrophoresis) from a contaminant having DNA-dependent ATP hydrolysis activity. Concentration of the LexA repressor was determined using a molar extinction coefficient of 7300 M−1 cm−1 at 280 nm (25Schnarr M. Daune M. FEBS Lett. 1984; 171: 207-210Google Scholar). Lactate dehydrogenase and pyruvate kinase were both purchased from Sigma as ammonium sulfate suspensions; working solutions were prepared by centrifuging a homogeneous aliquot and resuspending the protein pellet in reaction buffer. Single- and double-stranded DNA from bacteriophage M13mp7 and plasmid DNA from pBR322 were purified according to procedures outlined by Messing (26Messing J. Methods Enzymol. 1983; 101: 20-78Google Scholar); duplex DNA from M13 bacteriophage replicative form (“homologous”) and pBR322 (“nonhomologous”) were linearized using EcoRI and NdeI restriction endonucleases, respectively. Molar nucleotide concentrations were determined using extinction coefficients of 8780 M−1 cm−1 for ssDNA and 6500 M−1 cm−1 for dsDNA, both at 260 nm. Etheno M13 DNA was prepared from viral DNA as described (27Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Google Scholar), while poly(dT) was purchased from Pharmacia Biotech, Inc. and dissolved as a concentrated stock using TE buffer (10 mM Tris-HCl and 1 mM EDTA (pH 7.5)); nucleotide concentrations were determined using molar extinction coefficients of 7000 and 8520 M−1 cm−1 at 260 nm, respectively. Unless otherwise indicated, incubations and reactions were conducted at 37°C in a standard buffer comprised of 25 mM Tris-hydrochloride (pH 7.5), 10 mM magnesium chloride, 50 mM sodium chloride, 0.1 mM dithiothreitol, and 1 mM ATP using the following order of addition: to standard buffer with the indicated amounts of magnesium chloride, sodium chloride, and nucleotide triphosphate cofactor (ATP, dATP, or ATPγS), and a regenerating system consisting of 8 mM phosphoenolpyruvate (Sigma) and 12.5 units/ml pyruvate kinase (25 units/ml for dATP), ssDNA (M13, poly(dT) or etheno M13), and RecA protein were incubated. When present, the SSB protein (0.25 μM) was added 5 min after the RecA protein, unless otherwise noted. All reactions were initiated with the addition of 10 μM LexA repressor. If included, either homologous or nonhomologous linear dsDNA was added immediately prior to the LexA repressor. Cleavage of the LexA repressor by RecA protein was measured using 15% SDS-polyacrylamide gel electrophoresis as described previously (12Little J.W. Edmiston S.H. Pacelli L.Z. Mount D.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3225-3229Google Scholar, 28Lavery P.E. Kowalczykowski S.C. J. Mol. Biol. 1988; 203: 861-874Google Scholar). The integrated optical density (IOD) of resolved bands corresponding to intact LexA repressor, the two proteolytic fragments, and the RecA protein were quantitated from Coomassie Brilliant Blue-stained gels using a Millipore Bioimage Imaging System. Lane-dependent artifacts due to gel loading or running were accounted for in a given reaction by normalizing the IOD values measured for both intact and proteolytic fragments of LexA repressor to that determined for the RecA protein. The extent of LexA repressor cleavage was calculated as a ratio of the normalized IOD value for the intact LexA repressor relative to the sum of each normalized IOD value for the intact LexA repressor and for the two proteolytic fragments. All extents of repressor cleavage are corrected for the amount of cleaved LexA repressor at zero time (≥85% intact). Cleavage rates were determined using the slope of a least squares fit of the initial linear portion of reaction time courses. The single-stranded DNA-dependent hydrolysis of ATP was measured using a continuous spectrophotometric assay that couples ADP production to the oxidation of NADH (29Kreuzer K.N. Jongeneel C.V. Methods Enzymol. 1983; 100: 144-160Google Scholar) as adapted for use with the RecA protein (30Kowalczykowski S.C. Krupp R.A. J. Mol. Biol. 1987; 193: 97-113Google Scholar). Assays were carried out under those indicated conditions used for measuring the rate of LexA proteolysis and included 0.2 mg/ml NADH (Sigma) and 12.5 units/ml lactate dehydrogenase. In reactions including repressor, the LexA repressor was added last to ongoing assays, and steady state rates of ATP hydrolysis were determined within regions of the time course where the LexA repressor remained at least 50% intact. Both the rates of LexA proteolysis and ATP hydrolysis reported are the average of minimally two independently determined sets of data. Experimental errors in the rates of ssDNA-dependent LexA repressor cleavage and ATP hydrolysis were calculated to be less than or equal to ±9 and ±5%, respectively, of the average values. In the presence of ATP, the binding of RecA protein to ssDNA is stabilized by magnesium ion (27Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Google Scholar). Moreover, characterization of the ssDNA-dependent ATP hydrolysis activity of the RecA protein demonstrated that the magnesium ion concentration affects the extent of RecA nucleoprotein filament formation (30Kowalczykowski S.C. Krupp R.A. J. Mol. Biol. 1987; 193: 97-113Google Scholar). Maximum rates of ATP hydrolysis are normally observed at a ratio of one RecA protein monomer per three nucleotides of ssDNA. However, secondary structure intrinsic to ssDNA is stabilized by elevated levels of magnesium ion and prohibits RecA protein from saturating the ssDNA (31Muniyappa K. Shaner S.L. Tsang S.S. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2757-2761Google Scholar); consequently, this results in apparently lower maximal rates of ATP hydrolysis and higher binding stoichiometries (30Kowalczykowski S.C. Krupp R.A. J. Mol. Biol. 1987; 193: 97-113Google Scholar). For this reason, we examined the influence of magnesium ion on LexA repressor cleavage as a function of RecA protein concentration at a fixed amount of M13 ssDNA (Fig. 1). Over an initial range of RecA protein concentration (<0.75 μM), the rates of LexA repressor cleavage are equivalent at either 1 or 10 mM magnesium chloride and increase linearly with respect to the amount of RecA protein. At 10 mM magnesium chloride, proteolytic activity saturates at 0.8 μM/min, yielding an apparent site size of approximately five nucleotides of ssDNA per monomer of RecA protein. However, at 1 mM magnesium chloride, the level at which LexA repressor cleavage plateaus is enhanced (∼1.35 μM/min) and the apparent stoichiometry is decreased to three to four nucleotides/RecA protein monomer. Both of these observations are consistent with the idea that destabilization of secondary structure at reduced magnesium chloride concentrations allows RecA protein to access more ssDNA, thereby resulting in a greater extent of ternary complex. Furthermore, the apparent first order rate constant for LexA proteolysis, as determined from the data at sub-saturating RecA protein concentrations, is virtually the same (kcat∼0.92 min−1) at either 1 or 10 mM magnesium chloride, confirming that the activity directly reflects the amount of active RecA protein ternary complex formed. In support of the critical requirement for ssDNA in activation of RecA protein during SOS induction, the rates of LexA repressor proteolysis were approximately 10-fold lower in the presence of either linear M13 or pBR322 dsDNA (data not shown). Due to a preferential affinity for ssDNA, SSB protein disrupts secondary structure within native ssDNA and, upon being subsequently displaced, allows RecA protein to polymerize on these normally inaccessible regions (30Kowalczykowski S.C. Krupp R.A. J. Mol. Biol. 1987; 193: 97-113Google Scholar, 31Muniyappa K. Shaner S.L. Tsang S.S. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2757-2761Google Scholar, 32Morrical S.W. Lee J. Cox M.M. Biochemistry. 1986; 25: 1482-1494Google Scholar). Thus, despite being a competitor for limited binding sites, the SSB protein indirectly contributes to the formation of a contiguous RecA nucleoprotein filament on ssDNA (30Kowalczykowski S.C. Krupp R.A. J. Mol. Biol. 1987; 193: 97-113Google Scholar, 33Kowalczykowski S.C. Clow J.C. Somani R. Varghese A. J. Mol. Biol. 1987; 193: 81-95Google Scholar). As illustrated in Fig. 2A (dashed lines), this effect of the SSB protein is manifest as a stimulation in the ATP hydrolysis activity of the RecA protein when M13 ssDNA is used as a substrate at elevated magnesium ion concentrations (28Lavery P.E. Kowalczykowski S.C. J. Mol. Biol. 1988; 203: 861-874Google Scholar, 30Kowalczykowski S.C. Krupp R.A. J. Mol. Biol. 1987; 193: 97-113Google Scholar). Before binding sites on M13 ssDNA become limiting, the steady-state rate of ATP hydrolysis increases linearly with RecA protein concentration in a manner unaffected by SSB protein; however, at saturating concentrations of RecA protein, the maximal rate of ATP hydrolysis achieved in the presence of SSB protein (∼21 μM/min) is approximately 2-fold greater than that observed in its absence (∼10 μM/min). In quantitative agreement with the enhanced rate at which ATP hydrolysis plateaus, there is a corresponding decrease in the apparent ssDNA binding stoichiometry derived for the RecA protein from ∼six to ∼three nucleotides of ssDNA per protein monomer. These findings argue that in the presence of SSB protein more of the M13 substrate is available to support the ssDNA-dependent ATP hydrolysis activity of RecA protein. To assess the effect of SSB protein on LexA repressor digestion, proteolysis was examined under conditions identical to those used for ATP hydrolysis. As observed for ATP hydrolysis (Fig. 2A), inclusion of SSB protein enhances the rate at which proteolytic cleavage saturates (∼1.15 μM/min) relative to reactions in which it was omitted (∼0.65 μM/min) (Fig. 2B). Furthermore, the need for 1.5- to 2-fold higher RecA protein concentrations to achieve maximal rates of both LexA repressor cleavage and ATP hydrolysis in the presence of SSB protein indicates that the SSB protein enables the RecA protein to utilize the native ssDNA more completely. These results are consistent with the interpretation that by removing normally inaccessible regions of secondary structure from native ssDNA, SSB protein facilitates formation of the RecA protein ternary complex and, consequently, stimulates those biochemical activities dependent on it (i.e. ATP hydrolysis, LexA repressor cleavage). However, in contrast to ATP hydrolysis, LexA proteolysis is attenuated at sub-stoichiometric concentrations of RecA protein (≤0.50 μM) in reactions containing SSB protein (Fig. 2B). To determine the origin of this reduction in cleavage activity, the hydrolysis of ATP was also examined in the presence of LexA repressor (Fig. 2A, solid lines). Addition of the LexA repressor to reactions containing SSB protein results in a decrease in ATP hydrolysis at less than stoichiometric concentrations of the RecA protein, similar to that observed in the proteolysis assay. This inhibition of RecA protein activity is SSB protein-dependent as the LexA repressor has no effect on ATP hydrolysis in the absence of SSB protein (Fig. 2A). Furthermore, optimal inhibition of the ssDNA-dependent activities of the RecA protein caused by the LexA repressor requires saturating amounts of SSB protein (i.e. either reducing the SSB protein concentration or increasing the M13 ssDNA concentration diminished the relative amount of inhibition, data not shown). Collectively, these observations indicate not only that the observed inhibition is a direct result of RecA protein being supplanted from the ssDNA by SSB protein but that the LexA repressor acts in an auxiliary manner to bring about this replacement of the RecA protein-ATP-ssDNA complex. Similar to most protein-nucleic acid complexes (34Record Jr., M.T. Anderson C.F. Lohman T.M. Q. Rev. Biophys. 1978; 11: 103-178Google Scholar), the stability of RecA protein-ssDNA complexes decreases with increasing salt concentrations (27Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Google Scholar). However, this sensitivity to disruption by salt is not due to competitive binding effects associated with cation displacement from the ssDNA phosphate backbone but rather is the result of anion displacement from the RecA protein during complex formation (35Menetski J.P. Varghese A. Kowalczykowski S.C. J. Biol. Chem. 1992; 267: 10400-10404Google Scholar). Consequently, interactions between LexA and RecA proteins may destabilize the RecA protein-ATP-ssDNA ternary complex and thus account for the repressor-dependent increase in the inhibition of RecA protein activities by SSB protein. Since ATP hydrolysis and LexA proteolysis require formation of the same ternary complex, to address this possibility the salt sensitivities of these ssDNA-dependent activities of RecA protein were examined in parallel. As shown in Fig. 3, increasing amounts of sodium chloride cause a decrease in the ability of the RecA protein to promote either the hydrolysis of ATP or the cleavage of LexA repressor, in both the absence and presence of SSB protein. The concentrations of sodium chloride resulting in 50% inhibition of ATP hydrolysis and LexA proteolysis in the absence of SSB protein are similar (150 and 170 mM, respectively); in assays containing SSB protein, 250 mM sodium chloride is necessary for this degree of inhibition of hydrolysis, compared with 225 mM for proteolysis. In the case of either activities, SSB protein increases the apparent salt resistance by enhancing the formation of a more complete nucleoprotein filament. Furthermore, the amounts of sodium chloride required to reduce ATP hydrolysis by RecA protein to half-maximal levels, in either the absence or presence of SSB protein, are relatively unaffected by LexA repressor (Fig. 3A). Thus, based on the comparable salt sensitivities for these M13 ssDNA-dependent activities of the RecA protein, the LexA repressor does not drastically alter the steady-state stability of the RecA protein-ATP-ssDNA complex. The stability and structure of the RecA nucleoprotein complexes are modulated by interaction with nucleotide cofactors. Binding of ATP increases the equilibrium affinity of the RecA protein for ssDNA (27Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Google Scholar) and yields an extended conformation of the RecA protein-ssDNA complex (36Stasiak A. Egelman E.H. Kucherlapati R. Smith G.R. Genetic Recombination. American Society for Microbiology, Washington, D. C.1988: 265Google Scholar, 37Heuser J. Griffith J. J. Mol. Biol. 1989; 210: 473-484Google Scholar, 38DiCapua E. Schnarr M. Ruigrok R.W. Lindner P. Timmins P.A. J. Mol. Biol. 1990; 214: 557-570Google Scholar); besides being fundamental to certain enzymatic activities, the high affinity ssDNA-binding state induced by ATP is required for RecA protein to be able to compete with, and displace, the SSB protein (9Kowalczykowski S.C. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 539-575Google Scholar). In order to investigate the effects of repressor on the interaction of RecA protein with nucleotide cofactors, and to compare the requirements for nucleotide in ATP hydrolysis and LexA proteolysis, each of these ssDNA-dependent activities was examined as a function of the nucleoside triphosphate concentration (Fig. 4). As has been shown by others (39Weinstock G.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 8829-8834Google Scholar, 40Kowalczykowski S.C. Biochemistry. 1986; 25: 5872-5881Google Scholar, 41Menetski J.P. Varghese A. Kowalczykowski S.C. Biochemistry. 1988; 27: 1205-1212Google Scholar), the ATP hydrolysis activity of RecA protein is sigmoid with respect to nucleotide concentration. The ATP concentration needed to achieve half-maximal rates of hydrolysis defines an apparent Km of 120 ± 4 μM for the RecA protein (Fig. 4A), which is similar to previously reported values (39Weinstock G.M. McEntee K. Lehman I.R. J. Biol. Chem. 1981; 256: 8829-8834Google Scholar, 40Kowalczykowski S.C. Biochemistry. 1986; 25: 5872-5881Google Scholar, 41Menetski J.P. Varghese A. Kowalczykowski S.C. Biochemistry. 1988; 27: 1205-1212Google Scholar). While maximal rates of ATP hydrolysis attained are unchanged, the presence of the LexA repressor shifts the apparent Km of the RecA protein to a slightly higher ATP concentration (160 ± 8 μM). Furthermore, inclusion of repressor partially inhibits the M13 ssDNA-dependent ATP hydrolysis activity of the RecA protein at ATP concentrations between 60 and 200 μM (Fig. 4A). In agreement with earlier characterizations (6Roberts J.W. Roberts C.W. Craig N.L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4714-4718Google Scholar, 10Craig N.L. Roberts J.W. Nature. 1980; 283: 26-29Google Scholar, 12Little J.W. Edmiston S.H. Pacelli L.Z. Mount D.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3225-3229Google Scholar, 42Craig N.L. Roberts J.W. J. Biol. Chem. 1981; 256: 8039-8044Google Scholar, 43Phizicky E.M. Roberts J.W. Cell. 1981; 25: 259-267Google Scholar, 44Weinstock G.M. McEntee K. J. Biol. Chem. 1981; 256: 10883-10888Google Scholar), ATP, dATP, and the essentially non-hydrolyzable analogue ATPγS support formation of RecA protein ternary complex that is active in proteolysis (Fig. 4B). Th" @default.
• W2004642972 created "2016-06-24" @default.
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• W2004642972 date "1996-09-01" @default.
• W2004642972 modified "2023-10-14" @default.
• W2004642972 title "Interaction of Escherichia coli RecA Protein with LexA Repressor" @default.
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