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. 1990 Jan;10(1):113–119. doi: 10.1128/mcb.10.1.113

Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells.

F L Lin 1, K Sperle 1, N Sternberg 1
PMCID: PMC360718  PMID: 2294397

Abstract

To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.

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Selected References

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

  1. Anderson R. A., Eliason S. L. Recombination of homologous DNA fragments transfected into mammalian cells occurs predominantly by terminal pairing. Mol Cell Biol. 1986 Sep;6(9):3246–3252. doi: 10.1128/mcb.6.9.3246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. A., Kato S., Camerini-Otero R. D. A pattern of partially homologous recombination in mouse L cells. Proc Natl Acad Sci U S A. 1984 Jan;81(1):206–210. doi: 10.1073/pnas.81.1.206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brenner D. A., Smigocki A. C., Camerini-Otero R. D. Double-strand gap repair results in homologous recombination in mouse L cells. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1762–1766. doi: 10.1073/pnas.83.6.1762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brenner D. A., Smigocki A. C., Camerini-Otero R. D. Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells. Mol Cell Biol. 1985 Apr;5(4):684–691. doi: 10.1128/mcb.5.4.684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brouillette S., Chartrand P. Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions. Mol Cell Biol. 1987 Jun;7(6):2248–2255. doi: 10.1128/mcb.7.6.2248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cesarone C. F., Bolognesi C., Santi L. Improved microfluorometric DNA determination in biological material using 33258 Hoechst. Anal Biochem. 1979 Nov 15;100(1):188–197. doi: 10.1016/0003-2697(79)90131-3. [DOI] [PubMed] [Google Scholar]
  7. Chakrabarti S., Seidman M. M. Intramolecular recombination between transfected repeated sequences in mammalian cells is nonconservative. Mol Cell Biol. 1986 Jul;6(7):2520–2526. doi: 10.1128/mcb.6.7.2520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doetschman T., Gregg R. G., Maeda N., Hooper M. L., Melton D. W., Thompson S., Smithies O. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987 Dec 10;330(6148):576–578. doi: 10.1038/330576a0. [DOI] [PubMed] [Google Scholar]
  9. Folger K. R., Thomas K., Capecchi M. R. Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei. Mol Cell Biol. 1985 Jan;5(1):59–69. doi: 10.1128/mcb.5.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jasin M., de Villiers J., Weber F., Schaffner W. High frequency of homologous recombination in mammalian cells between endogenous and introduced SV40 genomes. Cell. 1985 Dec;43(3 Pt 2):695–703. doi: 10.1016/0092-8674(85)90242-9. [DOI] [PubMed] [Google Scholar]
  11. Kucherlapati R. S., Eves E. M., Song K. Y., Morse B. S., Smithies O. Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA. Proc Natl Acad Sci U S A. 1984 May;81(10):3153–3157. doi: 10.1073/pnas.81.10.3153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lin F. L., Sperle K., Sternberg N. Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products. Mol Cell Biol. 1990 Jan;10(1):103–112. doi: 10.1128/mcb.10.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lin F. L., Sperle K., Sternberg N. Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process. Mol Cell Biol. 1984 Jun;4(6):1020–1034. doi: 10.1128/mcb.4.6.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McKnight S. L. Functional relationships between transcriptional control signals of the thymidine kinase gene of herpes simplex virus. Cell. 1982 Dec;31(2 Pt 1):355–365. doi: 10.1016/0092-8674(82)90129-5. [DOI] [PubMed] [Google Scholar]
  15. Nicholls R. D., Fischel-Ghodsian N., Higgs D. R. Recombination at the human alpha-globin gene cluster: sequence features and topological constraints. Cell. 1987 May 8;49(3):369–378. doi: 10.1016/0092-8674(87)90289-3. [DOI] [PubMed] [Google Scholar]
  16. Rubnitz J., Subramani S. The minimum amount of homology required for homologous recombination in mammalian cells. Mol Cell Biol. 1984 Nov;4(11):2253–2258. doi: 10.1128/mcb.4.11.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Seidman M. M. Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative. Mol Cell Biol. 1987 Oct;7(10):3561–3565. doi: 10.1128/mcb.7.10.3561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Singh H., Bieker J. J., Dumas L. B. Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors. Gene. 1982 Dec;20(3):441–449. doi: 10.1016/0378-1119(82)90213-x. [DOI] [PubMed] [Google Scholar]
  19. Smithies O., Gregg R. G., Boggs S. S., Koralewski M. A., Kucherlapati R. S. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature. 1985 Sep 19;317(6034):230–234. doi: 10.1038/317230a0. [DOI] [PubMed] [Google Scholar]
  20. Song K. Y., Chekuri L., Rauth S., Ehrlich S., Kucherlapati R. Effect of double-strand breaks on homologous recombination in mammalian cells and extracts. Mol Cell Biol. 1985 Dec;5(12):3331–3336. doi: 10.1128/mcb.5.12.3331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Song K. Y., Schwartz F., Maeda N., Smithies O., Kucherlapati R. Accurate modification of a chromosomal plasmid by homologous recombination in human cells. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6820–6824. doi: 10.1073/pnas.84.19.6820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Szostak J. W., Orr-Weaver T. L., Rothstein R. J., Stahl F. W. The double-strand-break repair model for recombination. Cell. 1983 May;33(1):25–35. doi: 10.1016/0092-8674(83)90331-8. [DOI] [PubMed] [Google Scholar]
  24. Thomas K. R., Folger K. R., Capecchi M. R. High frequency targeting of genes to specific sites in the mammalian genome. Cell. 1986 Feb 14;44(3):419–428. doi: 10.1016/0092-8674(86)90463-0. [DOI] [PubMed] [Google Scholar]
  25. Wagner M. J., Sharp J. A., Summers W. C. Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1441–1445. doi: 10.1073/pnas.78.3.1441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wake C. T., Vernaleone F., Wilson J. H. Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells. Mol Cell Biol. 1985 Aug;5(8):2080–2089. doi: 10.1128/mcb.5.8.2080. [DOI] [PMC free article] [PubMed] [Google Scholar]

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