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• W2046402644 abstract "We have recently obtained evidence for a direct linkage between the S0.5 (S0.5 is the substrate concentration required for half-maximal velocity) value of a nucleoside triphosphate and the conformational state of the RecA-ssDNA complex, with an S0.5 value of 125 µM or less required for stabilization of the strand exchange-active conformation. For example, although ATP and ITP are hydrolyzed by the RecA protein with the same turnover number (18 min−1), ATP (S0.5 = 45 µM) functions as a cofactor for the strand exchange reaction, whereas ITP (S0.5 = 500 µM) is inactive as a strand exchange cofactor. The RecA protein crystal structure suggests that cofactor specificity is determined by Asp100, which likely forms a hydrogen bond with the exocyclic 6-amino group of ATP; the higher S0.5 value for ITP is presumably due to unfavorable interactions between Asp100 and the 6-carbonyl group of the inosine ring. To test this hypothesis, we prepared a mutant RecA protein in which Asp100 was replaced by an asparagine residue. The S0.5(ITP) for the [D100N]RecA protein is 125 µM, indicating favorable interactions between the Asn100 side chain and the 6-carbonyl group of ITP. Correspondingly, ITP functions as a cofactor for the strand exchange activity of the [D100N]RecA protein. This result demonstrates the importance of the residue at position 100 in determining nucleotide cofactor specificity and underscores the importance of the S0.5 value in the RecA protein-promoted strand exchange reaction. We have recently obtained evidence for a direct linkage between the S0.5 (S0.5 is the substrate concentration required for half-maximal velocity) value of a nucleoside triphosphate and the conformational state of the RecA-ssDNA complex, with an S0.5 value of 125 µM or less required for stabilization of the strand exchange-active conformation. For example, although ATP and ITP are hydrolyzed by the RecA protein with the same turnover number (18 min−1), ATP (S0.5 = 45 µM) functions as a cofactor for the strand exchange reaction, whereas ITP (S0.5 = 500 µM) is inactive as a strand exchange cofactor. The RecA protein crystal structure suggests that cofactor specificity is determined by Asp100, which likely forms a hydrogen bond with the exocyclic 6-amino group of ATP; the higher S0.5 value for ITP is presumably due to unfavorable interactions between Asp100 and the 6-carbonyl group of the inosine ring. To test this hypothesis, we prepared a mutant RecA protein in which Asp100 was replaced by an asparagine residue. The S0.5(ITP) for the [D100N]RecA protein is 125 µM, indicating favorable interactions between the Asn100 side chain and the 6-carbonyl group of ITP. Correspondingly, ITP functions as a cofactor for the strand exchange activity of the [D100N]RecA protein. This result demonstrates the importance of the residue at position 100 in determining nucleotide cofactor specificity and underscores the importance of the S0.5 value in the RecA protein-promoted strand exchange reaction. The RecA protein of Escherichia coli (Mr 37,842, 352 amino acids) is essential for homologous genetic recombination and for the postreplicative repair of damaged DNA. The purified RecA protein will promote a variety of DNA pairing reactions that presumably reflect in vivo recombination functions. The most extensively investigated DNA pairing activity is the ATP-dependent three-strand exchange reaction, in which a circular ssDNA 1The abbreviations used are: ssDNAsingle-stranded DNAdsDNAdouble-stranded DNAφXbacteriophage φX174SSBE. coli single-stranded DNA-binding proteinPCRpolymerase chain reactionATPγSadenosine 5′-O-(thiotriphosphate). molecule and a homologous linear dsDNA molecule are recombined to yield a nicked circular dsDNA molecule and a linear ssDNA molecule. This reaction proceeds in three phases. In the first phase, the circular ssDNA substrate is coated with RecA protein to form a presynaptic complex; this complex will catalyze the hydrolysis of ATP to ADP and Pi. In the second phase, the presynaptic complex interacts with a dsDNA molecule, the homologous sequences are brought into register, and pairing between the circular ssDNA and the complementary strand from the dsDNA is initiated. In the third phase, the complementary linear strand is completely transferred to the circular ssDNA by unidirectional branch migration to yield the nicked circular dsDNA and displaced linear ssDNA products (Roca and Cox, 9Roca A.I. Cox M.M. Crit. Rev. Biochem. Mol. Biol. 1990; 25: 415-456Crossref PubMed Scopus (362) Google Scholar; Kowalczykowski et al., 6Kowalczykowski S.C. Dixon D.A. Eggleston A.K. Lauder S.D. Rehrauer W.M. Microbiol. Rev. 1994; 58: 401-465Crossref PubMed Google Scholar). single-stranded DNA double-stranded DNA bacteriophage φX174 E. coli single-stranded DNA-binding protein polymerase chain reaction adenosine 5′-O-(thiotriphosphate). The presynaptic complex formed between RecA protein and ssDNA is the active recombinational entity in the strand exchange reaction. The RecA protein binds cooperatively to ssDNA, forming a right-handed helical protein filament with one RecA monomer per four nucleotides of ssDNA and six RecA monomers per turn of the filament. In the absence of nucleotide cofactor or in the presence of ADP, the helical filament adopts a “collapsed” or “closed” conformation (helical pitch: 65 Å) that is inactive in strand exchange. In the presence of ATP or the nonhydrolyzable ATP analog, ATPγS, however, the filament assumes an “extended” or “open” conformation (helical pitch: 95 Å) that is active in strand exchange (Egelman, 4Egelman E.H. Curr. Opin. Struct. Biol. 1993; 3: 189-197Crossref Scopus (63) Google Scholar). We have been examining the mechanism of the nucleotide cofactor-mediated isomerization of the RecA-ssDNA complex and have identified a linkage between the S0.5 value 2S0.5 is the substrate concentration required for half-maximal velocity. of a nucleoside triphosphate and the conformational state of the RecA-ssDNA complex (Menge and Bryant, 8Menge K.L. Bryant F.R. Biochemistry. 1992; 31: 5151-5157Crossref PubMed Scopus (20) Google Scholar; Meah and Bryant, 7Meah Y.S. Bryant F.R. J. Biol. Chem. 1993; 268: 23991-23996Abstract Full Text PDF PubMed Google Scholar; Stole and Bryant, 10Stole E. Bryant F.R. J. Biol. Chem. 1994; 269: 7919-7925Abstract Full Text PDF PubMed Google Scholar, 11Stole E. Bryant F.R. J. Biol. Chem. 1995; 270: 20322-20328Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). These studies have shown that a nucleoside triphosphate must have an S0.5 value of 100-120 µM or lower in order to stabilize the strand exchange-active conformation of the RecA-ssDNA complex. For example, although ATP and ITP are hydrolyzed by the RecA protein with identical turnover numbers (18 min−1), ATP (S0.5 = 45 µM) functions as a cofactor for the strand exchange reaction, whereas ITP (S0.5 = 500 µM) is inactive as a strand exchange cofactor. The x-ray crystal structure of the RecA protein-ADP complex indicates that cofactor specificity is determined by Asp100, which forms a hydrogen bond with the exocyclic 6-amino group of adenosine base (Story et al., 12Story R.M. Weber I.T. Steitz T.A. Nature. 1992; 355: 318-325Crossref PubMed Scopus (676) Google Scholar); it seems likely that a similar contact is made with ATP in the RecA-ssDNA-ATP complex (Fig. 1), although no structural information is available for this complex. The higher S0.5 value for ITP, relative to that for ATP, is presumably due to unfavorable interactions between the negatively charged Asp100 side chain and the 6-carbonyl group of the inosine ring. To test this hypothesis, we prepared a mutant RecA protein in which Asp100 was replaced by an asparagine residue. The effect of this mutation on the nucleotide cofactor specificity of the RecA protein is described in this report. Wild-type RecA protein was prepared as described previously (Cotterill et al., 2Cotterill S.M. Satterthwait A.C. Fersht A.R. Biochemistry. 1982; 21: 4332-4337Crossref PubMed Scopus (57) Google Scholar). ATP and ITP were from Sigma. [α-32P]ATP and [γ-32P]ATP were from ICN. [γ-32P]ITP was prepared from IDP using [γ-32P]ATP and nucleoside diphosphate kinase (Sigma) as described previously (Menge and Bryant, 8Menge K.L. Bryant F.R. Biochemistry. 1992; 31: 5151-5157Crossref PubMed Scopus (20) Google Scholar). E. coli SSB was from Pharmacia Biotech Inc.. Circular φX ssDNA ((+)-strand) and circular φX dsDNA were from New England Biolabs; linear φX dsDNA was prepared from circular φX dsDNA as described (Cox and Lehman, 3Cox M.M. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3433-3437Crossref PubMed Scopus (222) Google Scholar). Single- and double-stranded DNA concentrations were determined by absorbance at 260 nm using the conversion factors 36 and 50 µg/ml/A260, respectively. All DNA concentrations are expressed as total nucleotides. The mutant [D100N]RecA gene was produced by the polymerase chain reaction (PCR)-based overlap extension method essentially as described (Ho et al., 5Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar). The template for RecA mutagenesis consisted of a pUC19 vector containing a 1300-base pair E. coli DNA fragment carrying the wild-type recA gene and promoter cloned into a BamHI/HindIII site; the mutagenesis primers were 5′-CACGCGCTGCCAATCTACG-3′ and 5′-CGTAGATTGGCAGCGCGTG-3′ (the codon for Asn100 is underlined and the nucleotide mismatch is in bold). The resulting PCR fragment containing the mutant RecA gene and promoter region was cloned into pUC19 to yield the plasmid, pUCrecA(D100N). The entire [D100N]RecA gene and promoter region was sequenced to confirm that only the desired change had been introduced during the mutagenesis procedure. pUCrecA(D100N) was then introduced into the RecA deletion strain, BNN124, and the mutant [D100N]RecA protein was expressed and purified by methods that have been described previously (Bryant, 1Bryant F.R. J. Biol. Chem. 1988; 263: 8716-8723Abstract Full Text PDF PubMed Google Scholar). In an effort to reengineer the nucleotide cofactor specificity of the RecA protein, we prepared a new mutant RecA protein in which aspartic acid 100 was replaced by an asparagine residue. The expectation was that the D100N mutation would allow the protein to form hydrogen bonding interactions with the 6-carbonyl group of ITP that are not possible in the wild-type protein (Fig. 1). If the new interactions resulted in a significant decrease in the S0.5 value for ITP, we predicted that the mutation would convert the RecA protein into an ITP-activated DNA recombinase. The purified [D100N]RecA protein is shown in Fig. 2. The ssDNA-dependent hydrolysis of ATP and ITP by the wild-type and [D100N]RecA protein was analyzed under standard reaction conditions (pH 7.5, 37°C). The dependence of the rate of ssDNA-dependent NTP hydrolysis on NTP concentration is shown in Fig. 3, and the steady-state kinetic parameters for the hydrolysis of each NTP are presented in Table I.Table IKinetic parameters for wild-type and [D100N]RecA protein-catalyzed NTP hydrolysis (pH 7.5)NTPVmax/[Et]S0.5nHmin−1µMWild typeATP18453ITP195003D100NATP18853ITP181253 Open table in a new tab The turnover number (Vmax/[Et]) for ssDNA-dependent ATP hydrolysis by the [D100N]RecA protein was 18 min−1, a value identical to that obtained for the wild-type RecA protein. The S0.5(ATP) for the [D100N]RecA protein was 85 µM, approximately 2-fold higher than the value of 45 µM for the wild-type RecA protein. The ATP saturation curves were sigmoidal and identical Hill coefficients (nH) of 3 were obtained for both proteins, indicating that both proteins are subject to positive cooperativity with respect to ATP concentration. These results show that the D100N mutation has a minimal effect on the steady-state kinetics of ssDNA-dependent ATP hydrolysis by the RecA protein. The turnover number for ssDNA-dependent ITP hydrolysis by the [D100N]RecA protein was also 18 min−1, a value again equivalent to that obtained for the wild-type protein. Moreover, the ITP saturation curves were sigmoidal and identical Hill coefficients of 3 were obtained for both proteins, indicating that both proteins are subject to positive cooperativity with respect to ITP concentration. However, the S0.5(ITP) for the [D100N]RecA protein was 125 µM compared with a value of 500 µM for the wild-type protein. Thus, the D100N mutation results in a 4-fold decrease in the S0.5(ITP), presumably due to more favorable interactions of the asparagine side chain with the 6-carbonyl group of the inosine ring. The three-strand exchange activity of the [D100N]RecA protein was evaluated under standard reaction conditions (pH 7.5, 37°C). In the three-strand exchange assay, a circular φX ssDNA molecule and a linear φX dsDNA molecule are recombined to form a nicked circular dsDNA molecule and a linear ssDNA molecule; the substrates and products of this reaction are readily monitored by agarose gel electrophoresis (Cox and Lehman, 3Cox M.M. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3433-3437Crossref PubMed Scopus (222) Google Scholar). As shown in Fig. 4, the wild-type RecA protein has full strand exchange activity in the presence of ATP (3 mM), but has no detectable activity in the presence of ITP (3 mM), consistent with our previous results (Menge and Bryant, 8Menge K.L. Bryant F.R. Biochemistry. 1992; 31: 5151-5157Crossref PubMed Scopus (20) Google Scholar). In contrast, the [D100N]RecA protein exhibited full strand exchange activity in the presence of either ATP or ITP (3 mM). Thus, the D100N mutation converts the RecA protein into an ITP-activated DNA recombinase. Our results show that it is possible to alter the nucleoside triphosphate cofactor specificity of the RecA protein by mutating the aspartic acid residue at position 100 of the RecA polypeptide. This finding confirms that the hydrogen bonding interaction between Asp100 and the 6-amino group of the adenosine ring that is apparent in the crystal structure of the RecA-ADP complex (which presumably represents the strand exchange-inactive closed conformation of the protein) also contributes to nucleotide cofactor binding in the strand exchange-active open conformation of the RecA-ssDNA complex." @default.
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• W2046402644 title "Reengineering the Nucleotide Cofactor Specificity of the RecA Protein by Mutation of Aspartic Acid 100" @default.
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