Abstract
Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264→Glu) and recA665 (Tyr264→His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD (1989) Molecular biology of the cell. Garland Publishing, New York
Bianchi ME, Radding CE (1983) Insertions, deletions and mismatches in heteroduplex DNA made by RecA protein. Cell 35:511–520
Burckhardt SE, Woodgate R, Sheuermann RH, Echols H (1988) The UmuD mutagenesis protein of Escherichia coli: overproduction, purification and cleavage by RecA. Proc Natl Acad Sci USA 85:1811–1815
Castellazzi M, George J, Buttin G (1972) Prophage induction and cell division in E. coli. I. Further characterization of the thermosensitive mutation tif-1 whose expression mimics the effects of UV irradiation. Mol Gen Genet 119:139–152
Cazaux C, Defais M (1992) Genetical and biochemical evidence for the involvement of the coprotease domain of Escherichia coli RecA protein in recombination. J Mol Biol 223:823–829
Cazaux C, Larminat F, Defais M (1991) Site-directed mutagenesis in the Escherichia coli recA gene. Biochimie 73:281–284
Churchward G, Belin D, Nagamine Y (1984) A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene 31:165–171
Cotterill SM, Satterthwait AC, Fersht AR (1982) RecA protein from Escherichia coli. A very rapid and simple purification procedure: binding of adenosine 5′-triphosphate and adenosine 5′-diphosphate by the homogeneous protein. Biochemistry 21:4332–4337
Cox MM, Lehman IR (1981) RecA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc Natl Acad Sci USA 78:3433–3437
Craig NL, Roberts JW (1981) Escherichia coli RecA protein-directed cleavage of phage lambda repressor requires polynucleotide. Nature 283:26–30
DasGupta C, Shibata T, Cunningham RP, Radding CM (1980) The topology of homologous pairing promoted by RecA protein. Cell 22:437–446
DiCapua E, Cuillel M, Hewat E, Schnarr M, Timmins PA, Ruigrok RWH (1992) Activation of RecA protein: the open helix model for LexA cleavage. J Mol Biol 226:707–719
Dutreix M, Moreau PL, Bailone A, Galibert F, Battista JR, Walker GR, Devoret R (1989) New RecA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV-mutagenesis. J Bacteriol 115:2415–2423
Egelman EH, Stasiak A (1988) Structure of helical RecA-DNA complexes. II. Local conformational changes visualized in bundles of RecA-ATP-γ-S filaments. J Mol Biol 200:329–349
Freitag NE, McEntee K (1991) Site-directed mutagenesis in the RecA protein of Escherichia coli. J Biol Chem 266:7058–7066
Kim JI, Cox MM, Inman RB (1992a) On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. I. Bypassing a short heterologous insert in one DNA substrate. J Biol Chem 267:16438–16443
Kim JI, Cox MM, Inman RB (1992b) On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. II. Four strand exchanges. J Biol Chem 267:16444–16449
Kirby EP, Jacob F, Goldthwait DA (1967) Prophage induction and filament formation in a mutant strain of Escherichia coli. Proc Natl Acad Sci USA 58:1903–1910
Knight KL, McEntee K (1985a) Affinity labeling of a tyrosine residue in the ATP binding site of the RecA protein from Escherichia coli with 5′-p-fluorosulfonylbenzoyladenosine. J Biol Chem 260:10177–10184
Knight KL, McEntee K (1985b) Covalent modification of the RecA protein from Escherichia coli with the photoaffinity label 8-azidoadenosine 5′-triphosphate. J Biol Chem 260:867–872
Knight KL, McEntee K (1985c) Tyrosine 264 in the RecA protein from Escherichia coli is the site of modification by the photoaffinity label 8-azido adenosine 5′-triphosphate. J Biol Chem 260:10185–10191
Knight KL, Aoki KH, Ujita EL, McEntee K (1984) Identification,1,- of the amino acid substitutions in two mutant forms of the RecA protein from Escherichia coli. J Biol Chem 259:11279–11283
Kobayashi N, Knight K, McEntee K (1987) Evidence for nucleotide mediated changes in the domain structure of the RecA protein in Escherichia coli. Biochemistry 26:6801–6810
Kowalczykowski SC (1991) Biochemistry of genetic recombination: energetics and mechanism of strand exchange. Annu Rev Biophys Chem 20:539–575
Larminat F, Cazaux C, Germanier M, Defais M (1992) New mutants in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activities. J Bacteriol 216:106–112
Lavery PE, Kowalczykowski SC (1988) Biochemical analysis of the temperature-inducible constitutive protease activity of the RecA441 protein of Escherichia coli. J Mol Biol 203:861–874
Lavery PE, Kowalczykowski SC (1992) Biochemical basis of the constitutive repressor cleavage activity of RecA730 protein. J Biol Chem 267:20648–20658
Little JW, Edminston SH, Pacelli LZ, Mount DW (1980) Cleavage of the Escherichia coli LexA protein by the RecA protease. Proc Natl Acad Sci USA 78:4199–4203
Livneh Z, Lehman IR (1982) Recombinational bypass of pyrimidine dimers promoted by the RecA protein of Escherichia coli. Proc Natl Acad Sci USA 79:3171–3175
Menetski JP, Kowalczykowski SC (1985) Interaction of RecA protein with single-stranded DNA. Quantitative aspects of binding affinity by nucleotide cofactors. J Mol Biol 185:281–295
Menetski JP, Bear DG, Kowalczykowski SC (1990) Stable DNA heteroduplex formation catalysed by the Escherichia coli RecA protein in the absence of ATP hydrolysis. Proc Natl Acad Sci USA 87:21–25
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York.
Moreau PL (1987) Effects of overproduction of single-stranded DNA-binding protein on RecA protein-dependent processes in Escherichia coli. J Mol Biol 194:621–643
Nohmi T, Battista JR, Dodson LA, Walker GC (1988) RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci USA 85:1816–1820
Ogawa T, Wabiko H, Tsurimoto T, Horii T, Masukata H, Ogawa H (1979) Characteristics of purified RecA protein and the regulation of its synthesis in vivo. Cold Spring Harbor Symp Quant Biol 43:909–915
Phizicky EM, Roberts JW (1981) Induction of SOS functions: regulation of proteolytic activity of E. coli RecA protein by interaction with DNA and nucleoside triphosphate. Cell 25:259–267
Roberts JW, Roberts CW, Craig NL, Phizicky EM (1979) Activity of the Escherichia coli recA-gene product. Cold Spring Harbour Symp Quant Biol 43:917–920
Roca AI, Cox MM (1990) The RecA protein: structure and function. CRC Crit Rev Biochem 25:415–456
Rosselli W, Stasiak A (1991) The ATPase activity of RecA protein is needed to push the DNA strand exchange through heterologous regions. EMBO J 10:4391–4396
Shinagawa H, Iwasaki H, Kato T, Nakata A (1988) RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci USA 85:1806–1810
Story RM, Steitz TA (1992) Structure of the RecA protein-ADP complex. Nature 355:374–376
Story RM, Weber IT, Steitz TA (1992) The structure of the E. coli RecA protein monomer and polymer. Nature 355:318–325
Taylor JW, Ott J, Eckstein F (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res 13:8764–8785
Walker GC (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 8:60–93
West SC (1992) Enzymes and molecular mechanisms of genetic recombination. Annu Rev Biochem 61:603–640
Witkin EM, McCall JO, Volkert MR, Wermundsen IE (1982) Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli. Mol Gen Genet 185:43–50
Author information
Authors and Affiliations
Additional information
Communicated by R. Devoret
Rights and permissions
About this article
Cite this article
Cazaux, C., Mazard, AM. & Defais, M. Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele. Molec. Gen. Genet. 240, 296–301 (1993). https://doi.org/10.1007/BF00277070
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00277070