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. 1978 Feb;75(2):620–624. doi: 10.1073/pnas.75.2.620

Homology-dependent cutting in trans of DNA in extracts of Escherichia coli: an approach to the enzymology of genetic recombination.

E Cassuto, J Mursalim, P Howard-Flanders
PMCID: PMC411307  PMID: 345273

Abstract

An in vitro system is described in which the cutting of crosslinked phiX replicative form (RF) I DNA molecules by the uvr system of Escherichia coli induces the cutting of homologous undamaged DNA during incubation with crude extracts of thermally induced E. coli (lambda precA+) lysogens. This reaction, which has also been observed in intact E. coli lysogens infected with lambda phages, is dependent on the presence of functional recA+ and uvrB+ gene products. Extracts from thermally induced lambda precA+ lysogens of E. coli proved to be substantially more active than extracts from nonlysogenic cells of the same strain. The results provide preliminary evidence for an endonuclease activity that cuts intact superhelical DNA in response to interaction with homologus damaged DNA. In the present paper, we describe an in vitro system in which both the endonucleolytic cutting of DNA containing crosslinks and the induced cutting of undamaged DNA can be studied without purification of the participating enzymes. Although the information obtained is fragmentary and often puzzling, we feel that this system can contribute to an understanding of the complex mechanisms involved in repair and recombination.

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Selected References

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

  1. Benbow R. M., Krauss M. R. Recombinant DNA formation in a cell-free system from Xenopus laevis eggs. Cell. 1977 Sep;12(1):191–204. doi: 10.1016/0092-8674(77)90197-0. [DOI] [PubMed] [Google Scholar]
  2. Benbow R. M., Zuccarelli A. J., Sinsheimer R. L. Recombinant DNA molecules of bacteriophage phi chi174. Proc Natl Acad Sci U S A. 1975 Jan;72(1):235–239. doi: 10.1073/pnas.72.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brooks K., Clark A. J. Behavior of lambda bacteriophage in a recombination deficienct strain of Escherichia coli. J Virol. 1967 Apr;1(2):283–293. doi: 10.1128/jvi.1.2.283-293.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. CLARK A. J., MARGULIES A. D. ISOLATION AND CHARACTERIZATION OF RECOMBINATION-DEFICIENT MUTANTS OF ESCHERICHIA COLI K12. Proc Natl Acad Sci U S A. 1965 Feb;53:451–459. doi: 10.1073/pnas.53.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cassuto E., Gross N., Bardwell E., Howard-Flanders P. Genetic effects of photoadducts and photocross-links in the DNA of phage lambda exposed to 360 nm light and tri-methylpsoralen or khellin. Biochim Biophys Acta. 1977 Apr 19;475(4):589–600. doi: 10.1016/0005-2787(77)90319-7. [DOI] [PubMed] [Google Scholar]
  6. Cole R. S., Levitan D., Sinden R. R. Removal of psoralen interstrand cross-links from DNA of Escherichia coli: mechanism and genetic control. J Mol Biol. 1976 May 5;103(1):39–59. doi: 10.1016/0022-2836(76)90051-6. [DOI] [PubMed] [Google Scholar]
  7. Cole R. S. Repair of DNA containing interstrand crosslinks in Escherichia coli: sequential excision and recombination. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1064–1068. doi: 10.1073/pnas.70.4.1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cole R. S., Zusman D. Sedimentation properties of phage DNA molecules containing light-induced psoralen cross-links. Biochim Biophys Acta. 1970 Dec 14;224(2):660–662. doi: 10.1016/0005-2787(70)90607-6. [DOI] [PubMed] [Google Scholar]
  9. Defais M., Fauquet P., Radman M., Errera M. Ultraviolet reactivation and ultraviolet mutagenesis of lambda in different genetic systems. Virology. 1971 Feb;43(2):495–503. doi: 10.1016/0042-6822(71)90321-7. [DOI] [PubMed] [Google Scholar]
  10. Epstein W., Kim B. S. Potassium transport loci in Escherichia coli K-12. J Bacteriol. 1971 Nov;108(2):639–644. doi: 10.1128/jb.108.2.639-644.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fox M. S. On the mechanism of integration of transforming deoxyribonucleate. J Gen Physiol. 1966 Jul;49(6):183–196. doi: 10.1085/jgp.49.6.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Gottesman S., Gottesman M. Excision of prophage lambda in a cell-free system. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2188–2192. doi: 10.1073/pnas.72.6.2188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gudas L. J. The induction of protein X in DNA repair and cell division mutants of Escherichia coli. J Mol Biol. 1976 Jul 5;104(3):567–587. doi: 10.1016/0022-2836(76)90121-2. [DOI] [PubMed] [Google Scholar]
  15. Holloman W. K., Radding C. M. Recombination promoted by superhelical DNA and the recA gene of Escherichia coli. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3910–3914. doi: 10.1073/pnas.73.11.3910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Holloman W. K., Wiegand R., Hoessli C., Radding C. M. Uptake of homologous single-stranded fragments by superhelical DNA: a possible mechanism for initiation of genetic recombination. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2394–2398. doi: 10.1073/pnas.72.6.2394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Howard-Flanders P., Lin P. F. Genetic recombination induced by DNA cross-links in repressed phage lambda. Genetics. 1973 Apr;73(Suppl):85–90. [PubMed] [Google Scholar]
  18. Howard-Flanders P., Theriot L. Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. Genetics. 1966 Jun;53(6):1137–1150. doi: 10.1093/genetics/53.6.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Inouye M. Pleiotropic effect of the rec A gene of Escherichia coli: uncoupling of cell division from deoxyribonucleic acid replication. J Bacteriol. 1971 May;106(2):539–542. doi: 10.1128/jb.106.2.539-542.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. JACOB F., WOLLMAN E. L. Etude génétique d'un bactériophage tempéré d'Escherichia coli. III. Effet du rayonnement ultraviolet sur la recombinaison génétique. Ann Inst Pasteur (Paris) 1955 Jun;88(6):724–749. [PubMed] [Google Scholar]
  21. Kohn K. W., Steigbigel N. H., Spears C. L. Cross-linking and repair of DNA in sensitive and resistant strains of E. coli treated with nitrogen mustard. Proc Natl Acad Sci U S A. 1965 May;53(5):1154–1161. doi: 10.1073/pnas.53.5.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kushner S. R., Nagaishi H., Templin A., Clark A. J. Genetic recombination in Escherichia coli: the role of exonuclease I. Proc Natl Acad Sci U S A. 1971 Apr;68(4):824–827. doi: 10.1073/pnas.68.4.824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. McEntee K., Hesse J. E., Epstein W. Identification and radiochemical purification of the recA protein of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3979–3983. doi: 10.1073/pnas.73.11.3979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Miura A., Tomizawa J. I. Studies on radiation-sensitive mutants of E. coli. 3. Participation of the rec system in induction of mutation by ultraviolet irradiation. Mol Gen Genet. 1968;103(1):1–10. doi: 10.1007/BF00271151. [DOI] [PubMed] [Google Scholar]
  27. Morpurgo G. Induction of Mitotic Crossing-over in Aspergillus Nidulans by Bifunctional Alkylating Agents. Genetics. 1963 Sep;48(9):1259–1263. doi: 10.1093/genetics/48.9.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ross P., Howard-Flanders P. Initiation of recA+-dependent recombination in Escherichia coli (lambda). I. Undamaged covalent circular lambda DNA molecules in uvrA+ recA+ lysogenic host cells are cut following superinfection with psoralen-damaged lambda phages. J Mol Biol. 1977 Nov 25;117(1):137–158. doi: 10.1016/0022-2836(77)90028-6. [DOI] [PubMed] [Google Scholar]
  29. Ross P., Howard-Flanders P. Initiation of recA+-dependent recombination in Escherichia coli (lambda). II. Specificity in the induction of recombination and strand cutting in undamaged covalent circular bacteriophage 186 and lambda DNA molecules in phage-infected cells. J Mol Biol. 1977 Nov 25;117(1):159–174. doi: 10.1016/0022-2836(77)90029-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Sadowski P. D., Vetter D. Genetic recombination of bacteriophage T7 DNA in vitro. Proc Natl Acad Sci U S A. 1976 Mar;73(3):692–696. doi: 10.1073/pnas.73.3.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spring S. B., Maassab H. F., Kendal A. P., Murphy B. R., Chanock R. M. Cold-adapted variants of influenza virus A. I. Comparison of the genetic properties of ts mutants and five cold-adapted variants of influenza virus A. Virology. 1977 Mar;77(1):337–343. doi: 10.1016/0042-6822(77)90430-5. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Wickner W., Brutlag D., Schekman R., Kornberg A. RNA synthesis initiates in vitro conversion of M13 DNA to its replicative form. Proc Natl Acad Sci U S A. 1972 Apr;69(4):965–969. doi: 10.1073/pnas.69.4.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Witkin E. M. The mutability toward ultraviolet light of recombination-deficient strains of Escherichia coli. Mutat Res. 1969 Jul-Aug;8(1):9–14. doi: 10.1016/0027-5107(69)90135-3. [DOI] [PubMed] [Google Scholar]
  36. Witkin E. M. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev. 1976 Dec;40(4):869–907. doi: 10.1128/br.40.4.869-907.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]

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