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• W2037222730 abstract "The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoprotein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament. The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoprotein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament. Homologous DNA recombination is a vital component of DNA metabolism, central to processes such as recombinational DNA repair of stalled replication forks and the exchange of genetic material during meiosis in eukaryotes and conjugation in prokaryotes. RecA protein is the central recombinase in Escherichia coli, and RecA homologues are present in nearly every organism. In E. coli, RecA participates not only in the restart of stalled replication forks but also the induction of the SOS response upon cellular DNA damage distress and translesion DNA synthesis via the error-prone DNA polymerase V (1Lusetti S.L. Cox M.M. Annu. Rev. Biochem. 2002; 71: 71-100Crossref PubMed Scopus (353) Google Scholar, 2Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (871) Google Scholar, 3Schlacher K. Leslie K. Wyman C. Woodgate R. Cox M.M. Goodman M.F. Mol. Cell. 2005; 17: 561-572Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). RecA protein functions as a nucleoprotein filament. When bound to DNA, RecA promotes the hydrolysis of ATP or dATP. Hydrolysis occurs uniformly throughout the filament. ATP hydrolysis is important for some RecA processes including net disassembly of the nucleoprotein filament, bypass of heterologous insertions during DNA three-strand exchange, complete DNA strand exchange with DNA substrates longer than ∼3 kbp, and strand exchange with four DNA strands (1Lusetti S.L. Cox M.M. Annu. Rev. Biochem. 2002; 71: 71-100Crossref PubMed Scopus (353) Google Scholar, 4Jain S.K. Cox M.M. Inman R.B. J. Biol. Chem. 1994; 269: 20653-20661Abstract Full Text PDF PubMed Google Scholar, 5Kim J.I. Cox M.M. Inman R.B. J. Biol. Chem. 1992; 267: 16444-16449Abstract Full Text PDF PubMed Google Scholar, 6Kim J.I. Cox M.M. Inman R.B. J. Biol. Chem. 1992; 267: 16438-16443Abstract Full Text PDF PubMed Google Scholar, 7Shan Q. Cox M.M. Inman R.B. J. Biol. Chem. 1996; 271: 5712-5724Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 8Cox M.M. Annu. Rev. Microbiol. 2003; 57: 551-577Crossref PubMed Scopus (172) Google Scholar). The ATP hydrolytic cycles between adjacent subunits in the RecA filament bound to double-stranded DNA (dsDNA) 2The abbreviations used are: dsDNA, double-stranded DNA; ATPγS, adenosine 5′-O-(3-thiotriphosphate); PEP, phosphoenolpyruvate; SSB, single-stranded DNA-binding protein of Escherichia coli; ssDNA, single-stranded DNA; GAP, GTPase-activating protein; ADPNP, 5′-adenylyl-β,γ-imidodiphosphate. are coordinated such that waves of hydrolysis move sequentially through the filament with a separation of six subunits (9Cox J.M. Tsodikov O.V. Cox M.M. PLoS Biol. 2005; 3: 231-243Crossref Scopus (62) Google Scholar). RecA could potentially use the organized waves of ATP hydrolysis to act as a motor, driving completion of strand exchange beyond the barriers mentioned above, or to promote replication fork regression (10Robu M.E. Inman R.B. Cox M.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8211-8218Crossref PubMed Scopus (128) Google Scholar, 11Robu M.E. Inman R.B. Cox M.M. J. Biol. Chem. 2004; 279: 10973-10981Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The detailed mechanism by which the ATP hydrolysis is coordinated in the RecA-dsDNA nucleoprotein filament is currently unknown. RecA is a member of an ATPase family with characteristic single or multiple RecA-like folds, composed of a central β-sheet flanked by α-helices. The ATPase family includes helicases like PcrA, ABC transporters, and with relatively less structural similarity, the AAA+ family of ATPases including ClpA (12Ye J. Osborne A.R. Groll M. Rapoport T.A. Biochim. Biophys. Acta. 2004; 1659: 1-18Crossref PubMed Scopus (115) Google Scholar). These proteins all use the energy from ATP hydrolysis to move macromolecules or move along macromolecules, and hence, may be considered molecular motors. A sequence alignment of RecA and helicases including DnaB (13Leipe D.D. Aravind L. Grishin N.V. Koonin E.V. Genome Res. 2000; 10: 5-16PubMed Google Scholar) highlights the similarities. The authors call attention to a new motif, the [KR]×[KR] motif. This motif is conserved among the DnaB, RecA, Sms, and KaiC families, although absent from the archaean or eukaryotic homologues of RecA. The [KR]×[KR] motif is involved in the in trans catalysis of ATP hydrolysis for some RecA homologues including gp4 of bacteriophage T7 (13Leipe D.D. Aravind L. Grishin N.V. Koonin E.V. Genome Res. 2000; 10: 5-16PubMed Google Scholar, 14Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 15Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 16Crampton D.J. Guo S. Johnson D.E. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4373-4378Crossref PubMed Scopus (46) Google Scholar). The helicase domain of T7 gp4 constitutes a 5′ to 3′ hexameric ring helicase. The crystal structure of a gp4 fragment that retains hexamer formation and helicase activities (residues 241-566) (15Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar) strays from 6-fold rotational symmetry. Indeed, only four of six subunits have bound nucleotide (ADPNP). Arg522 is the third residue in the [KR]×[KR] motif, and in the four subunits that bind ADPNP, Arg522 is close (∼3.5 Å) to the γ-phosphate of the bound nucleotide of the neighboring subunit. However, Arg522 is displaced in the two nucleotide-free subunits to more than 10 Å away from the equivalent position where the nucleotide would be bound. A conformational change such as this is suggested to couple hydrolysis and helicase activity. It is proposed that Arg522 of T7 gp4 senses hydrolysis of ATP, communicates the hydrolysis between subunits, and promotes conformational changes associated with ATP hydrolysis (14Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 15Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 16Crampton D.J. Guo S. Johnson D.E. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4373-4378Crossref PubMed Scopus (46) Google Scholar). In this manner, Arg522 would mimic an arginine finger, as is found in Ras and its GTPase-activating protein (GAP). In the Ras-GAP system, it is postulated that Arg789 of GAP similarly reaches across the GAP-Ras interface to stabilize the γ-phosphate of the Ras-bound ATP (17Scheffzek K. Lautwein A. Kabsch W. Reza Ahmadian M. Wittinghofer A. Nature. 1996; 384: 591-596Crossref PubMed Scopus (144) Google Scholar). Another example of in trans catalysis is the RuvB protein (18Putnam C.D. Clancy S.B. Tsuruta H. Gonzalez S. Wetmur J.G. Tainer J.A. J. Mol. Biol. 2001; 311: 297-310Crossref PubMed Scopus (146) Google Scholar, 19Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar, 20Hishida T. Han Y.-W. Fujimoto S. Iwasaki H. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9573-9577Crossref PubMed Scopus (35) Google Scholar). The RuvB protein is an ATPase that forms a hexameric ring and complexes with RuvA to promote branch migration that is dependent on ATP hydrolysis. Although this protein exhibits some structural similarity to RecA, it appears to lack the [KR]×[KR] motif. The crystal structure features a dimer of Thermus thermophilus RuvB, with each subunit having three domains (labeled N, M, and C). The nucleotide is bound between the N and M domains (19Yamada K. Kunishima N. Mayanagi K. Ohnishi T. Nishino T. Iwasaki H. Shinagawa H. Morikawa K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1442-1447Crossref PubMed Scopus (83) Google Scholar). The structure of each subunit is very similar, except for the area immediately around the bound nucleotide. One subunit appears to have a triphosphate bound, while the other appears to lack a γ-phosphate; the authors postulated that the structural differences between the subunits could reflect conformational changes dependent on ATP hydrolysis. The crystal structures of wild type and several point mutants of Thermatoga maritima RuvB also indicate ADP is bound between two domains of the RuvB subunit (18Putnam C.D. Clancy S.B. Tsuruta H. Gonzalez S. Wetmur J.G. Tainer J.A. J. Mol. Biol. 2001; 311: 297-310Crossref PubMed Scopus (146) Google Scholar), suggesting to the authors that the state of the bound nucleotide guides conformational changes between RuvB subunits. More specifically, sensor 1, Walker A, and Walker B were proposed to sense the γ-phosphate and divalent cation, while sensor 2 would accommodate the sugar ring and diphosphate. A study of E. coli RuvB point mutants further extends the proposition that ATP hydrolysis causes conformational changes between subunits by indicating that Arg174 (corresponding to Arg170 of T. maritima RuvB) acts in trans across the subunit interface in the RuvB hexamer (20Hishida T. Han Y.-W. Fujimoto S. Iwasaki H. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9573-9577Crossref PubMed Scopus (35) Google Scholar). The Arg174 residue of RuvB does not comprise the active site for ATP hydrolysis in one subunit, but Arg174 is near the catalytic site of the adjacent subunit in the hexamer. The Walker A motif mutant K68A and R174A mutant are both deficient in ATP hydrolysis, but they were found to complement each other when mixed together (20Hishida T. Han Y.-W. Fujimoto S. Iwasaki H. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9573-9577Crossref PubMed Scopus (35) Google Scholar). Hence, Arg174 of RuvB appears to participate in trans in ATP hydrolysis. In E. coli RecA protein, the residues in the [KR]×[KR] motif are Lys248 and Lys250 (13Leipe D.D. Aravind L. Grishin N.V. Koonin E.V. Genome Res. 2000; 10: 5-16PubMed Google Scholar). These two residues are hypothesized to be important for ATP hydrolysis and potentially communicate ATP hydrolysis between adjacent subunits in the RecA nucleoprotein filament according to the similarities to homologous proteins described above. Furthermore, the promising importance of one or both of the Lys248 and Lys250 residues was reinforced upon examination of the structure of the RecA-dsDNA nucleoprotein filament (21VanLoock M.S. Yu X. Yang S. Lai A.L. Low C. Campbell M.J. Egelman E.H. Structure (Camb.). 2003; 11: 1-20Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). An impediment to the study of the RecA protein has been the lack of a crystal structure of the RecA filament bound to DNA. The crystal structure of the RecA filament with ADP bound (22Story R.M. Steitz T.A. Nature. 1992; 355: 374-376Crossref PubMed Scopus (560) Google Scholar) and the crystal structure of the RecA filament (23Story R.M. Weber I.T. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (680) Google Scholar) provided much insight. However, RecA is a DNA-dependent ATPase, and the apo-crystal represents the inactive form of the RecA filament (21VanLoock M.S. Yu X. Yang S. Lai A.L. Low C. Campbell M.J. Egelman E.H. Structure (Camb.). 2003; 11: 1-20Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Recently a reconstruction of the RecA filament with dsDNA, based on complexes viewed by electron microscopy, was resolved (21VanLoock M.S. Yu X. Yang S. Lai A.L. Low C. Campbell M.J. Egelman E.H. Structure (Camb.). 2003; 11: 1-20Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The crystal structure of the RecA filament was fit within electron microscopic images of the RecA-dsDNA filament. To fit the crystal structure electron density into the electron microscopy images of the nucleoprotein filament, the RecA core was rotated and resulted in several significant structural changes. One difference between the apo-crystal structure and the electron microscopic reconstruction of the RecA-dsDNA nucleoprotein filament is location of the ATP binding site (Fig. 1). The nucleotide is bound away from the subunit-subunit interface within the filament in the apo-crystal structure. In contrast, the nucleotide is bound in between adjacent subunits in the RecA-dsDNA nucleoprotein filament reconstruction. The location of the nucleotide between two RecA subunits is supported by recent crystal structures of the archaeal RadA filament (24Wu Y. He Y. Moya I.A. Qian X.G. Luo Y. Mol. Cell. 2004; 15: 423-435Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and the eukaryotic Rad51 filament (25Conway A.B. Lynch T.W. Zhang Y. Fortin G.S. Fung C.W. Symington L.S. Rice P.A. Nat. Struct. Mol. Biol. 2004; 11: 791-796Crossref PubMed Scopus (239) Google Scholar). VanLoock et al. (21VanLoock M.S. Yu X. Yang S. Lai A.L. Low C. Campbell M.J. Egelman E.H. Structure (Camb.). 2003; 11: 1-20Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) states there are several residues that do not contact the nucleotide in the apo-crystal structure but are near the nucleotide of the adjacent subunit in the electron microscopic reconstruction. The shifted residues include Lys216, Phe217, Arg222, Lys248, Lys250, and Pro254. The two residues Lys248 and Lys250 are of particular interest because, as noted above, they are the two residues that comprise the [KR]×[KR] motif in the E. coli RecA protein. In this study, we sought to examine the K248 residue as an introduction to studying potential roles for the [KR]×[KR] motif in E. coli RecA. The K248A mutation (26Nguyen T.T. Muench K.A. Bryant F.R. J. Biol. Chem. 1993; 268: 3107-3113Abstract Full Text PDF PubMed Google Scholar) has been previously examined, and although the K248A mutant protein appears to fold normally, the K248A mutant does not form a filament. As such, we chose to introduce a more subtle mutation: K248R. We find that the K248R mutant does bind DNA and forms a nucleoprotein filament, although it hydrolyzes neither ATP nor dATP at a readily measured rate. However, we discovered that a combination of the K248R mutant and another ATP hydrolysis-deficient mutant (27Campbell M.J. Davis R.W. J. Mol. Biol. 1999; 286: 417-435Crossref PubMed Scopus (37) Google Scholar, 28Campbell M.J. Davis R.W. J. Mol. Biol. 1999; 286: 437-445Crossref PubMed Scopus (37) Google Scholar), E96D, partially restores dATP hydrolysis. In the nucleoprotein filament, the Lys248 and Glu96 residues are located at the subunit-subunit interface near the bound nucleotide but are opposed across the interface. Direct or indirect coordination between these two residues could be a means of communicating ATP hydrolysis-mediated conformational changes between adjacent subunits in the RecA nucleoprotein filament. Enzymes and Biochemicals—The E. coli SSB protein was purified as described previously (7Shan Q. Cox M.M. Inman R.B. J. Biol. Chem. 1996; 271: 5712-5724Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The E. coli wild type RecA protein was purified as described previously (29Lusetti S.L. Wood E.A. Fleming C.D. Modica M.J. Korth J. Abbott L. Dwyer D.W. Roca A.I. Inman R.B. Cox M.M. J. Biol. Chem. 2003; 278: 16372-16380Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The RecA K248R mutant protein was purified in the same manner as wild type. The RecA K248R/E96D double mutant protein was purified in the same manner as wild type with the following changes: the protein was eluted twice from the ammonium sulfate pellet in a solution of R Buffer (20 mm Tris-Cl buffer (80% cation, pH 7.5), 10% glycerol (w/v), 1 mm dithiothreitol) plus 150 mm ammonium sulfate. The K248R/E96D mutant protein was eluted from the DEAE-Sepharose column with a linear gradient from R Buffer plus 100 mm KCl to R Buffer plus 500 mm KCl. The K248R/E96D mutant protein was eluted from the hydroxyapatite column with a linear gradient from 20 mm phosphate buffer (10 mm KH2PO4,10mm K2HPO4, 10% glycerol (w/v), 1 mm dithiothreitol) to 500 mm phosphate buffer (250 mm KH2PO4, 250 mm K2HPO4, 10% glycerol (w/v), 1 mm dithiothreitol). Peak fractions were identified by SDS-PAGE, pooled and dialyzed against R Buffer, and then loaded onto a PBE-94 column (Amersham Biosciences). The K248R/E96D mutant protein was eluted with a linear gradient from R Buffer to R Buffer plus 1 m KCl. The peak and nuclease-free fractions were pooled and concentrated with an Amicon Centricon concentrator (Millipore, Billerica, MA). The K248R/E96D mutant protein was dialyzed against R Buffer and flash-frozen in liquid nitrogen and finally stored at -80 °C. The E96D mutant RecA protein was purified similarly to the K248R/E96D mutant, excluding the PBE-94 column. The purified RecA protein (both mutants and wild type) and SSB protein concentrations were determined by absorbance at 280 nm, using extinction coefficients of ϵ280 = 2.23 × 104 m-1 cm-1(30Craig N.L. Roberts J.W. J. Biol. Chem. 1981; 256: 8039-8044Abstract Full Text PDF PubMed Google Scholar) and ϵ280 = 2.38 × 104 m-1 cm-1(31Lohman T.M. Overman L.B. J. Biol. Chem. 1985; 260: 3594-3603Abstract Full Text PDF PubMed Google Scholar), respectively. RecA protein and SSB preparations were free of detectable endo- and exonuclease activities on dsDNA and ssDNA. Unless otherwise noted, all reagents were purchased from Fisher. Phosphoenolpyruvate (PEP), pyruvate kinase, lactate dehydrogenase, phosphocreatine, ATP, dATP, and NADH were purchased from Sigma. Creatine kinase and ATPγS were purchased from Roche Applied Science. Restriction enzymes were purchased from New England Biolabs (Beverly, MA). Dithiothreitol was purchased from Research Organics (Cleveland, OH). DNA Substrates—Poly(dT) was purchased from Amersham Biosciences, and the approximate average length is 229 nucleotides. Bacteriophage ΦX174 circular ssDNA and replicative form I supercoiled circular duplex DNA were purchased from New England Biolabs and Invitrogen, respectively. Full-length linear duplex DNA (dsDNA) was generated by digesting ΦX174 replicative form I DNA (5386 bp) with the XhoI endonuclease. Circular single-stranded DNA from bacteriophage M13mp8 (7229 nucleotides) was prepared as described (32Neuendorf S.K. Cox M.M. J. Biol. Chem. 1986; 261: 8276-8282Abstract Full Text PDF PubMed Google Scholar). Concentrations of ssDNA and dsDNA were determined by absorbance using 108 and 151 μm A260-1, respectively, as conversion factors. All DNA concentrations are given in micromolar nucleotide concentrations. Circular Dichroism—Wild type RecA protein and two separate preparations of the RecA K248R/E96D double mutant protein were analyzed on a model Aviv 62A DS circular dichroism Spectrometer (Aviv, Lake-wood, NJ) equipped with a temperature controller. For each protein, the solution included 0.25 mg/ml protein, 10 mm KH2PO4, and 10 mm K2HPO4. The solutions were measured at 4 °C and 37 °C. Graphs of wavelength (nm) versus molar ellipticity (degree cm2 dmol-1 × 10-4) were normalized with respect to the absolute minimum at 208 nm. The RecA K248R/E96D protein CD spectrum and wild type RecA protein CD spectrum were superimposed to identify any differing points on the graphs. Two separate preparations of the RecA K248R/E96D mutant protein were examined. Reaction Conditions—All reactions were carried out at 37 °C in 25 mm Tris acetate buffer (80% cation, pH 7.5), 10 mm magnesium acetate, 5% (v/v) glycerol, 1 mm dithiothreitol, 3 mm potassium glutamate, 3 mm ATP or dATP, an ATP regenerating system (10 units/ml pyruvate kinase and 3 mm PEP), and concentrations of DNA and RecA protein were as described below and in the figure legends to Figs. 2 and 4, 5, 6, 7. The coupled spectrophotometric assay also contained 10 units/ml lactate dehydrogenase and 1.5 or 0.15 mm NADH. DNA and protein concentrations are indicated for each experiment. Reactions were incubated for 10 min before ATP or dATP was added to start the reaction.FIGURE 4A mixture of the K248R and E96D single mutant RecA proteins on poly(dT) hydrolyzes optimally at a ratio of 1 K248R:1 E96D. RecA-catalyzed dATP hydrolysis was monitored. Reactions included 8 μm total RecA protein, 6 μm poly(dT) linear ssDNA, and dATP. The assay was done in triplicate (all assays are shown). The optimal rate of dATP hydrolysis was observed at a 1:1 ratio of the two single mutants. This rate of dATP hydrolysis, 20.91 ± 0.08 μm/min, was ∼23% of the rate seen with wild type RecA protein alone under the same reaction conditions (91.54 ± 2.86 μm/min).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5The mixture of K248R+E96D mutants and the K248R/E96D double mutant RecA protein partially restores DNA-dependent ATP and dATP hydrolysis. RecA-catalyzed dATP or ATP hydrolysis was monitored. Reactions included 8 μm RecA protein, 6 μm poly(dT) linear ssDNA (when included), and dATP or ATP. The rates of hydrolysis were calculated for several trials with and without poly(dT) linear ssDNA for both ATP (A) and dATP (B) substrates. The average for each data set is plotted for each RecA protein. C, for each ATP or dATP data set, the rates of DNA-independent hydrolysis were averaged. The average rate of DNA-independent hydrolysis was then subtracted from each DNA-dependent rate of hydrolysis to result in the corrected rate of hydrolysis. The corrected rates of hydrolysis were then averaged and plotted for each RecA protein. Both E96D and K248R hydrolyzed neither ATP nor dATP at significant rates relative to wild type RecA protein. The 1:1 mixture of the K248R and E96D proteins hydrolyzed both ATP and dATP and hydrolyzed dATP at ∼25% of the wild type dATP hydrolysis rate. The K248R/E96D double mutant RecA protein also hydrolyzed both ATP and dATP, and the double mutant restored dATP hydrolysis to ∼8% of the wild type dATP hydrolysis rate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Wild type (WT) RecA and mutants form DNA-dependent nucleoprotein filaments. Electron micrographs show wild type and mutant RecA filaments on linear ssDNA. Reactions included 8 μm RecA protein and 6 μm poly(dT) linear ssDNA. The RecA protein was preincubated with the DNA for 10 min before dATP was added. The reaction was incubated for 10 min more to permit filament formation. Then ATPγS was added (to 3 mm) and the reaction incubated for 3 min to stabilize the filaments. Reactions were diluted 10-fold, except the K248R reaction (5-fold), before adhesion to the electron microscopy (alcians) grid. Nucleoprotein filaments were viewed with the electron microscope. For each protein, a total of at least 50 representative filaments were measured. Filaments shorter than 30 nm were difficult to distinguish from background and were not included in the reported totals. The results may thus underestimate the representation of shorter filament forms, especially for the K248R mutant. A histogram of filament length is shown for each protein next to the electron microscopy image. Wild type and E96D RecA proteins formed a range of nucleoprotein filament lengths with many long filaments greater than 600 nm. Since this is far longer than the available lengths of poly(dT), this implies an end-to-end joining of nucleoprotein filaments. The K248R and K248R/E96D mutant proteins only formed filaments with shorter lengths. The K248R filaments of measurable length were also present at a much reduced concentration. The E96D+K248R mixed filaments were a range of lengths, mostly median to the wild type and K248R filament lengths. All filaments are DNA-dependent, as no filaments were seen in the absence of DNA (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7The extending K248R/E96D mutant RecA protein filament is compromised and does not displace SSB. RecA-catalyzed dATP hydrolysis was monitored. Reactions included 8 μm RecA protein, 6 μm M13mp8 circular ssDNA, 0.6 μm SSB (when included), and dATP. The RecA protein was preincubated with the DNA for 10 min before dATP was added to initiate the reaction. Time 0 corresponds to the time of dATP addition. For both RecA proteins, there are three conditions: SSB was added initially with dATP (contiguous dashed line), SSB was added ∼20 min after dATP addition (solid line continued as dashed after SSB addition indicated by an arrow), or SSB storage buffer was added ∼20 min after dATP addition (continuous solid line). The top three curves represent hydrolysis by the wild type RecA protein; the bottom three curves represent hydrolysis by the K248R/E96D RecA protein. The solid curves represent dATP hydrolysis before SSB protein was added to the reaction; the dashed lines represent dATP hydrolysis after the addition of SSB protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ATP Hydrolysis Assays—A coupled spectrophotometric assay (33Lindsley J.E. Cox M.M. J. Biol. Chem. 1990; 265: 9043-9054Abstract Full Text PDF PubMed Google Scholar, 34Morrical S.W. Lee J. Cox M.M. Biochemistry. 1986; 25: 1482-1494Crossref PubMed Scopus (123) Google Scholar) was used to measure ATP and dATP hydrolysis by the RecA protein (29Lusetti S.L. Wood E.A. Fleming C.D. Modica M.J. Korth J. Abbott L. Dwyer D.W. Roca A.I. Inman R.B. Cox M.M. J. Biol. Chem. 2003; 278: 16372-16380Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The regeneration of ATP or dATP from ADP and PEP was coupled to the oxidation of NADH and monitored by the decrease in absorbance of NADH at 380 or 340 nm. The 380-nm wavelength was used instead of the absorption maximum at 340 nm so that the signal would remain within the linear range of the spectrophotometer for the duration of the experiment. However, the 340-nm wavelength was used instead of 380 nm to increase the sensitivity of the assay when the hydrolysis rate was slow. The assay was carried out using a Varian Cary 300 (Varian, Palo Alto, CA) dual beam spectrophotometer equipped with a temperature controller and 12-position cell changer. The cell path length and band pass were 1.0 cm and 2 nm, respectively. The NADH extinction coefficients at 380 nm of 1.21 mm-1 cm-1 and at 340 nm of 6.22 mm-1 cm-1 were used to calculate rates of ATP or dATP hydrolysis. DNA Three-strand Exchange Reactions—DNA three-strand exchange reactions were carried out at 37 °C in 25 mm Tris acetate buffer (80% cation, pH 7.5). RecA protein (up to 3 μm) was preincubated with ΦX174 circular ssDNA (up to 10 μm) for 10 min. SSB protein (to" @default.
• W2037222730 created "2016-06-24" @default.
• W2037222730 creator A5013311312 @default.
• W2037222730 creator A5020072322 @default.
• W2037222730 creator A5041118769 @default.
• W2037222730 creator A5080771015 @default.
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• W2037222730 date "2006-05-01" @default.
• W2037222730 modified "2023-10-11" @default.
• W2037222730 title "Complementation of One RecA Protein Point Mutation by Another" @default.
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