Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Jan;7(1):129–140. doi: 10.1128/mcb.7.1.129

Extrachromosomal recombination in mammalian cells as studied with single- and double-stranded DNA substrates.

F L Lin, K M Sperle, N L Sternberg
PMCID: PMC365049  PMID: 3561389

Abstract

We have previously proposed a model to account for the high levels of homologous recombination that can occur during the introduction of DNA into mammalian cells (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984). An essential feature of that model is that linear molecules with ends appropriately located between homologous DNA segments are efficient substrates for an exonuclease that acts in a 5'----3' direction. That process generates complementary single strands that pair in homologous regions to produce an intermediate that is processed efficiently to a recombinant molecule. An alternative model, in which strand degradation occurs in the 3'----5' direction, is also possible. In this report, we describe experiments that tested several of the essential features of the model. We first confirmed and extended our previous results with double-stranded DNA substrates containing truncated herpesvirus thymidine kinase (tk) genes (tk delta 5' and tk delta 3'). The results illustrate the importance of the location of double-strand breaks in the successful reconstruction of the tk gene by recombination. We next transformed cells with pairs of single-stranded DNAs containing truncated tk genes which should anneal in cells to generate the recombination intermediates predicted by the two alternative models. One of the intermediates would be the favored substrate in our original 5'----3' degradative model and the other would be the favored substrate in the alternative 3'----5' degradative model. Our results indicate that the intermediate favored by the 3'----5' model is 10 to 20 times more efficient in generating recombinant tk genes than is the other intermediate.

Full text

PDF
129

Images in this article

136Image
on p.136

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. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Broker T. R., Lehman I. R. Branched DNA molecules: intermediates in T4 recombination. J Mol Biol. 1971 Aug 28;60(1):131–149. doi: 10.1016/0022-2836(71)90453-0. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Cox M. M., Lehman I. R. Renaturation of DNA: a novel reaction of histones. Nucleic Acids Res. 1981 Jan 24;9(2):389–400. doi: 10.1093/nar/9.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Darby V., Blattner F. Homologous recombination catalyzed by mammalian cell extracts in vitro. Science. 1984 Dec 7;226(4679):1213–1215. doi: 10.1126/science.6334360. [DOI] [PubMed] [Google Scholar]
  9. Das S. K., Fujimura R. K. Mechanism of primer-template-dependent conversion of dNTP leads to dNMP by T5 DNA polymerase. J Biol Chem. 1980 Aug 10;255(15):7149–7154. [PubMed] [Google Scholar]
  10. Enquist L. W., Vande Woude G. F., Wagner M., Smiley J. R., Summers W. C. Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus. Gene. 1979 Nov;7(3-4):335–342. doi: 10.1016/0378-1119(79)90052-0. [DOI] [PubMed] [Google Scholar]
  11. Folger K. R., Wong E. A., Wahl G., Capecchi M. R. Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol. 1982 Nov;2(11):1372–1387. doi: 10.1128/mcb.2.11.1372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hines J. C., Ray D. S. Construction and characterization of new coliphage M13 cloning vectors. Gene. 1980 Nov;11(3-4):207–218. doi: 10.1016/0378-1119(80)90061-x. [DOI] [PubMed] [Google Scholar]
  13. Hsieh P., Meyn M. S., Camerini-Otero R. D. Partial purification and characterization of a recombinase from human cells. Cell. 1986 Mar 28;44(6):885–894. doi: 10.1016/0092-8674(86)90011-5. [DOI] [PubMed] [Google Scholar]
  14. Ishiura M., Hirose S., Uchida T., Hamada Y., Suzuki Y., Okada Y. Phage particle-mediated gene transfer to cultured mammalian cells. Mol Cell Biol. 1982 Jun;2(6):607–616. doi: 10.1128/mcb.2.6.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kenne K., Ljungquist S. A DNA-recombinogenic activity in human cells. Nucleic Acids Res. 1984 Apr 11;12(7):3057–3068. doi: 10.1093/nar/12.7.3057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Kucherlapati R. S., Spencer J., Moore P. D. Homologous recombination catalyzed by human cell extracts. Mol Cell Biol. 1985 Apr;5(4):714–720. doi: 10.1128/mcb.5.4.714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lai C. J., Nathans D. Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome. J Mol Biol. 1974 Oct 15;89(1):179–193. doi: 10.1016/0022-2836(74)90169-7. [DOI] [PubMed] [Google Scholar]
  19. Laird C. D. Chromatid structure: relationship between DNA content and nucleotide sequence diversity. Chromosoma. 1971 Mar 16;32(4):378–406. doi: 10.1007/BF00285251. [DOI] [PubMed] [Google Scholar]
  20. Lin F. L., Sperle K., Sternberg N. Homologous recombination in mouse L cells. Cold Spring Harb Symp Quant Biol. 1984;49:139–149. doi: 10.1101/sqb.1984.049.01.017. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Lin F. L., Sternberg N. Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome. Mol Cell Biol. 1984 May;4(5):852–861. doi: 10.1128/mcb.4.5.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  25. Miller C. K., Temin H. M. High-efficiency ligation and recombination of DNA fragments by vertebrate cells. Science. 1983 May 6;220(4597):606–609. doi: 10.1126/science.6301012. [DOI] [PubMed] [Google Scholar]
  26. Mizrahi V., Benkovic P., Benkovic S. J. Mechanism of DNA polymerase I: exonuclease/polymerase activity switch and DNA sequence dependence of pyrophosphorolysis and misincorporation reactions. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5769–5773. doi: 10.1073/pnas.83.16.5769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Potter H., Weir L., Leder P. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7161–7165. doi: 10.1073/pnas.81.22.7161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Riedel H. D., König H., Stahl H., Knippers R. Circular single stranded phage M13-DNA as a template for DNA synthesis in protein extracts from Xenopus laevis eggs: evidence for a eukaryotic DNA priming activity. Nucleic Acids Res. 1982 Sep 25;10(18):5621–5635. doi: 10.1093/nar/10.18.5621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rubnitz J., Subramani S. Rapid assay for extrachromosomal homologous recombination in monkey cells. Mol Cell Biol. 1985 Mar;5(3):529–537. doi: 10.1128/mcb.5.3.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shapira G., Stachelek J. L., Letsou A., Soodak L. K., Liskay R. M. Novel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4827–4831. doi: 10.1073/pnas.80.15.4827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Small J., Scangos G. Recombination during gene transfer into mouse cells can restore the function of deleted genes. Science. 1983 Jan 14;219(4581):174–176. doi: 10.1126/science.6294829. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. Wake C. T., Wilson J. H. Defined oligomeric SV40 DNA: a sensitive probe of general recombination in somatic cells. Cell. 1980 Aug;21(1):141–148. doi: 10.1016/0092-8674(80)90121-x. [DOI] [PubMed] [Google Scholar]
  38. Wake C. T., Wilson J. H. Simian virus 40 recombinants are produced at high frequency during infection with genetically mixed oligomeric DNA. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2876–2880. doi: 10.1073/pnas.76.6.2876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. de Saint Vincent B. R., Wahl G. M. Homologous recombination in mammalian cells mediates formation of a functional gene from two overlapping gene fragments. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2002–2006. doi: 10.1073/pnas.80.7.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES