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. 1980 May;77(5):2569–2573. doi: 10.1073/pnas.77.5.2569

Recognition of duplex DNA containing single-stranded regions by recA protein.

S C West, E Cassuto, J Mursalim, P Howard-Flanders
PMCID: PMC349443  PMID: 6446716

Abstract

Genetic recombination in Escherichia coli requires recA protein, the product of the recA+ gene. In this paper we show that purified recA protein, which binds strongly to denatured DNA, cooperatively recognizes DNA containing short single-stranded regions. The interaction of varying amounts of recA protein with DNA molecules was investigated by measuring its DNA-dependent ATPase activity. In 3mM Mg2+, the ATPase activity was stimulated by excess single-stranded DNA and was minimal with either intact circular or blunt-ended linear duplexes. Single-strand gaps of about 30 nucleotides were sufficient to increase the ATPase activity to a level almost as great as that observed with single-stranded DNA. Sedimentation studies at neutral pH showed cooperative binding of recA protein to single-stranded DNA or to duplex DNA containing single-stranded regions. In the presence of ATP, an intermediate rate of sedimentation was observed; in contrast, adenosine 5'-gamma-thiotriphosphate (ATP[S]) caused the formation of fast-sedimenting DNA-protein complexes. Gapped plasmid DNA plus recA protein and ATP[S] formed large aggregates containing thousands of molecules. Complex formation and stimulation of the ATPase activity of recA protein with duplex DNA containing single-stranded regions indicates that recA protein may change the conformation of the normally duplex molecules to a conformation prepared for homologous pairing.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Carter D. M., Radding C. M. The role of exonuclease and beta protein of phage lambda in genetic recombination. II. Substrate specificity and the mode of action of lambda exonuclease. J Biol Chem. 1971 Apr 25;246(8):2502–2512. [PubMed] [Google Scholar]
  2. Delius H., Mantell N. J., Alberts B. Characterization by electron microscopy of the complex formed between T4 bacteriophage gene 32-protein and DNA. J Mol Biol. 1972 Jun 28;67(3):341–350. doi: 10.1016/0022-2836(72)90454-8. [DOI] [PubMed] [Google Scholar]
  3. Emmerson P. T., West S. C. Identification of protein X of Escherichia coli as the recA+/tif+ gene product. Mol Gen Genet. 1977 Sep 21;155(1):77–85. doi: 10.1007/BF00268563. [DOI] [PubMed] [Google Scholar]
  4. Godson G. N. Evolution of phi-chi 174. Isolation of four new phi-chi-like phages and comparison with phi-chi 174. Virology. 1974 Mar;58(1):272–289. doi: 10.1016/0042-6822(74)90161-5. [DOI] [PubMed] [Google Scholar]
  5. Gudas L. J., Mount D. W. Identification of the recA (tif) gene product of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5280–5284. doi: 10.1073/pnas.74.12.5280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gudas L. J., Pardee A. B. Model for regulation of Escherichia coli DNA repair functions. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2330–2334. doi: 10.1073/pnas.72.6.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kikuchi Y., Nash H. A. Nicking-closing activity associated with bacteriophage lambda int gene product. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3760–3764. doi: 10.1073/pnas.76.8.3760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lin P. F., Bardwell E., Howard-Flanders P. Initiation of genetic exchanges in lambda phage--prophage crosses. Proc Natl Acad Sci U S A. 1977 Jan;74(1):291–295. doi: 10.1073/pnas.74.1.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. McEntee K. Genetic analysis of the Escherichia coli K-12 srl region. J Bacteriol. 1977 Dec;132(3):904–911. doi: 10.1128/jb.132.3.904-911.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McEntee K. Protein X is the product of the recA gene of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5275–5279. doi: 10.1073/pnas.74.12.5275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McEntee K., Weinstock G. M., Lehman I. R. Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2615–2619. doi: 10.1073/pnas.76.6.2615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McGavin S. Models of specifically paired like (homologous) nucleic acid structures. J Mol Biol. 1971 Jan 28;55(2):293–298. doi: 10.1016/0022-2836(71)90201-4. [DOI] [PubMed] [Google Scholar]
  13. Mount D. W. A mutant of Escherichia coli showing constitutive expression of the lysogenic induction and error-prone DNA repair pathways. Proc Natl Acad Sci U S A. 1977 Jan;74(1):300–304. doi: 10.1073/pnas.74.1.300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ogawa T., Wabiko H., Tsurimoto T., Horii T., Masukata H., Ogawa H. Characteristics of purified recA protein and the regulation of its synthesis in vivo. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):909–915. doi: 10.1101/sqb.1979.043.01.099. [DOI] [PubMed] [Google Scholar]
  15. Radding C. M. Genetic recombination: strand transfer and mismatch repair. Annu Rev Biochem. 1978;47:847–880. doi: 10.1146/annurev.bi.47.070178.004215. [DOI] [PubMed] [Google Scholar]
  16. Roberts J. W., Roberts C. W., Craig N. L., Phizicky E. M. Activity of the Escherichia coli recA-gene product. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):917–920. doi: 10.1101/sqb.1979.043.01.100. [DOI] [PubMed] [Google Scholar]
  17. Roberts J. W., Roberts C. W., Mount D. W. Inactivation and proteolytic cleavage of phage lambda repressor in vitro in an ATP-dependent reaction. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2283–2287. doi: 10.1073/pnas.74.6.2283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Roberts J. W., Roberts C. W. Proteolytic cleavage of bacteriophage lambda repressor in induction. Proc Natl Acad Sci U S A. 1975 Jan;72(1):147–151. doi: 10.1073/pnas.72.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rupp W. D., Howard-Flanders P. Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J Mol Biol. 1968 Jan 28;31(2):291–304. doi: 10.1016/0022-2836(68)90445-2. [DOI] [PubMed] [Google Scholar]
  20. Rupp W. D., Wilde C. E., 3rd, Reno D. L., Howard-Flanders P. Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli. J Mol Biol. 1971 Oct 14;61(1):25–44. doi: 10.1016/0022-2836(71)90204-x. [DOI] [PubMed] [Google Scholar]
  21. Sancar A., Rupp W. D. Physical map of the recA gene. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3144–3148. doi: 10.1073/pnas.76.7.3144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Scott J. F., Eisenberg S., Bertsch L. L., Kornberg A. A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication. Proc Natl Acad Sci U S A. 1977 Jan;74(1):193–197. doi: 10.1073/pnas.74.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shibata T., DasGupta C., Cunningham R. P., Radding C. M. Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1638–1642. doi: 10.1073/pnas.76.4.1638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sigal N., Delius H., Kornberg T., Gefter M. L., Alberts B. A DNA-unwinding protein isolated from Escherichia coli: its interaction with DNA and with DNA polymerases. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3537–3541. doi: 10.1073/pnas.69.12.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weinstock G. M., McEntee K., Lehman I. R. ATP-dependent renaturation of DNA catalyzed by the recA protein of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jan;76(1):126–130. doi: 10.1073/pnas.76.1.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. West S. C., Emmerson P. T. Induction of protein synthesis in Escherichia coli following UV- or gamma-irradiation, mitomycin C treatment or tif Expression. Mol Gen Genet. 1977 Feb 28;151(1):57–67. doi: 10.1007/BF00446913. [DOI] [PubMed] [Google Scholar]
  27. Wilson J. H. Nick-free formation of reciprocal heteroduplexes: a simple solution to the topological problem. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3641–3645. doi: 10.1073/pnas.76.8.3641. [DOI] [PMC free article] [PubMed] [Google Scholar]

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