Summary
Induction of prophage λ occurs in recA441 mutant lysogens after a shift to 42° C in the presence of adenine. If the synthesis of RecA441 protein is maintained at a low basal level by the presence of a second mutation in the recA441 gene, recA453, induction of prophage λ is prevented. The ability to induce prophage λ is restored by the introduction, on a transducing phage, of a second recA gene carrying the recA430 mutation; by itself, the RecA430 protein is devoid of activity against the λ repressor (Rebollo et al. 1984). In order to explain how the RecA430 protein might complement the RecA441 protein to provide λ repressor cleavage in a recA453-441 (recA430) diploid lysogen, we characterized the cleavage reaction catalysed by a mixture of these proteins in vitro. Our results suggest that, in the presence of dATP, the RecA441 and RecA430 proteins form mixed multimers on single-stranded DNA, in which the RecA441 protein molecules enhance the DNA binding affinity of RecA430 protein molecules, but RecA430 protein molecules support no cleavage of the λ repressor.
Although the effects of the RecA430 and single-strand binding (SSB) proteins are similar in vitro, we show that the SSB protein cannot substitute for the RecA430 protein in restoring λ repressor cleavage in a recA453-441 lysogen. Comparison of the stimulatory effect of long single-stranded DNA with that of (dA)14 oligonucleotides on the RecA441 protein-directed cleavage of the λ repressor in the presence of various nucleoside triphosphates (NTPs) indicates that the cooperative binding of the RecA441 protein to single-stranded DNA stabilizes the RecA protein-DNA complexes so that they remain intact long enough to support cleavage of the λ repressor. We conclude that the low basal level of the RecA441 protein in a recA453-441 cell is sufficient to cleave the λ repressor, under conditions where a normal basal level of RecA430 protein is also present allowing the formation of mixed multimers on single-stranded DNA regions normally present in the cell.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baluch J, Chase JW, Sussman R (1980) Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli. J Bacteriol 144:489–498
Brandsma JA, Stoorvogel J, van Sluis CA, van de Putte P (1982) Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Escherichia coli K-12. Gene 18:77–85
Burnette WN (1981) “Western Blotting” electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203
Castellazzi M, George J, Buttin G (1972) Prophage induction and cell division in E. coli. II. Linked (recA, zab) and unlinked (lex) suppressors of tif-1 mediated induction and filamentation. Mol Gen Genet 119:153–174
Chase JW, Whittier RF, Auerbach J, Sancar A, Rupp WD (1980) Amplification of single-strand DNA binding protein in Escherichia coli. Nucleic Acids Res 8:3215–3227
Chase JW, Murphy JB, Whittier RF, Lorensen E, Sninsky JJ (1983) Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli. J Mol Biol 163-164:193–211
Chrysogelos S, Register JC, Griffith J (1983) The structure of recA protein-DNA filaments. J Biol Chem 258:12624–12631
Cohen S, Knoll BJ, Little JW, Mount DW (1981) Preferential cleavage of phage λ repressor monomers by recA protease. Nature 294:182–184
Cox MM, Lehman IR (1982) recA protein-promoted DNA strand exchange. Stable complexes of recA protein and single-stranded DNA formed in the presence of ATP and single-stranded DNA binding protein. J Biol Chem 257:8523–8532
Craig NL, Roberts JW (1980) E. coli recA protein-directed cleavage of phage λ repressor requires polynucleotide. Nature 283:26–30
Craig NL, Roberts JW (1981) Function of nucleoside triphosphate and polynucleotide in Escherichia coli recA protein-directed cleavage of phage λ repressor. J Biol Chem 256:8039–8044
Devoret R, Pierre M, Moreau PL (1983) Prophage ø80 is induced in Escherichia coli K12 recA430. Mol Gen Genet 189:199–206
Dunn K, Chrysogelos S, Griffith J (1982) Electron microscopic visualization of RecA-DNA filaments. Evidence for a cyclic extension of duplex DNA. Cell 28:757–765
Flory J, Radding CM (1982) Visualization of RecA protein and its association with DNA: a priming effect of single-strand-binding protein. Cell 28:747–756
Gudas LJ, Mount DW (1977) Identification of the recA (tif) gene product of Escherichia coli. Proc Natl Acad Sci USA 74:5280–5284
Laemmli UK, Favre M (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 80:575–599
Lieberman HB, Witkin EM (1983) DNA degradation, UV sensitivity and SOS-mediated mutagenesis in strains of Escherichia coli deficient in single-strand DNA binding protein: Effects of mutations and treatments that alter levels of exonuclease V or RecA protein. Mol Gen Genet 190:92–100
Little JW, Edmiston SH, Pacelli LZ, Mount DW (1980) Cleavage of the Escherichia coli lexA protein by the recA protease. Proc Natl Acad Sci USA 77:3225–3229
Little JW, Mount DW (1982) The SOS regulatory system of Escherichia coli. Cell 29:11–22
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 250 and 366
McEntee K, Weinstock GM (1981) tif-1 mutation alters polynucleotide recognition by the recA protein of Escherichia coli. Proc Natl Acad Sci USA 78:6061–6065
McEntee K, Weinstock GM, Lehman IR (1981) Binding of the recA protein of Escherichia coli to single- and double-stranded DNA. J Biol Chem 256:8835–8844
Meyer RR, Voegele DW, Ruben SM, Rein DC, Trela JM (1982) Influence of single-stranded DNA binding protein on recA induction in Escherichia coli. Mutat Res 94:299–313
Moreau PL, Bailone A, Devoret R (1976) Prophage λ induction in Escherichia coli K12 envA uvrB: A highly sensitive test for potential carcinogens. Proc Natl Acad Sci USA 73:3700–3704
Moreau PL, Fanica M, Devoret R (1980) Induction of prophage λ does not require full induction of RecA protein synthesis. Biochimie 62:687–694
Moreau PL, Pélico JV, Devoret R (1982) Cleavage of λ repressor and synthesis of RecA protein induced by transferred UV-damaged F sex factor. Mol Gen Genet 186:170–179
Mount DW (1977) A mutant of Escherichia coli showing constitutive expression of the lysogenic induction and error-prone DNA repair pathways. Proc Natl Acad Sci USA 74:300–304
O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
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 (1980) Kinetics of RecA protein-directed inactivation of repressors of phage λ and phage P22. J Mol Biol 139:319–328
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
Quillardet P, Moreau PL, Ginsburg H, Mount DW, Devoret R (1982) Cell survival, UV-reactivation and induction of prophage λ in Escherichia coli K12 overproducing RecA protein. Mol Gen Genet 188:37–43
Radding CM (1982) Homologous pairing and strand exchange in genetic recombination. Annu Rev Genet 16:405–437
Rebollo JE, Moreau PL, Blanco M, Devoret R (1984) Restoration of RecA protein activity by genetic complementation. Mol Gen Genet 195:83–89
Resnick J, Sussman R (1982) Escherichia coli single-strand DNA binding protein from wild type and lexC113 mutant affects in vitro proteolytic cleavage of phage λ repressor. Proc Natl Acad Sci USA 79:2832–2835
Roberts JW, Roberts CW, Craig NL, Phizicky EM (1979) Activity of the Escherichia coli recA-gene product. Cold Spring Harbor Symp Quant Biol 43:917–920
Roberts JW, Roberts CW (1981) Two mutations that alter the regulatory activity of E. coli recA protein. Nature 290:422–424
Roberts JW, Phizicky EM, Burbee DG, Roberts CW, Moreau PL (1982) A brief consideration of the SOS inducing signal. Biochemie 64:805–807
Satta G, Gudas LJ, Pardee AB (1979) Degradation of Escherichia coli DNA: Evidence for limitation in vivo by protein X, the recA gene product. Mol Gen Genet 168:69–80
Soltis DA, Lehman IR (1983) recA protein promoted DNA strand exchange. J Biol Chem 258:6073–6077
Vales LD, Chase JW, Murphy JB (1980) Effect of ssbA1 and lexC113 mutations on lambda prophage induction, bacteriophage growth, and cell survival. J Bacteriol 143:887–896
Weinstock GM, McEntee K (1981) RecA protein-dependent proteolysis of bacteriophage λ repressor. Characterization of the reaction and stimulation by DNA-binding proteins. J Biol Chem 256:10883–10888
Whittier RF, Chase JW (1983) DNA repair properties of Escherichia coli tif-1, recAo281 and lexA1 strains deficient in single-strand DNA binding protein. Mol Gen Genet 190:101–111
Author information
Authors and Affiliations
Additional information
Communicated by H. Böhme
Rights and permissions
About this article
Cite this article
Moreau, P.L., Roberts, J.W. RecA protein — promoted λ repressor cleavage: complementation between RecA441 and RecA430 proteins in vitro. Molec Gen Genet 198, 25–34 (1984). https://doi.org/10.1007/BF00328696
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00328696