Abstract
Using as substrates, 1: the replicative form (RF) of phage M13 mp8 in which the reading frame of the lac Z' gene was disrupted by insertion of an octonucleotide, and 2: a restriction fragment one kb long, containing the functional lac Z' gene (isolated from wild type M13 mp8), we show that nuclear extracts from human cells (3 lines tested) promote the targeted replacement of the altered sequence by the functional one. Following incubation with the extracts, the DNA's were introduced in JM 109 bacteria (rec A- and lac Z'-) which were grown in presence of a colorimetric indicator of beta-galactosidase activity. Homologous recombination gives rise to the genotypical modification: lac Z'+ instead of lac Z'- in the bacteriophage DNA. This is revealed by phenotypical expression of the lac Z' gene product in replicating bacteriophage, i.e. the formation of blue instead of white plaques. The frequency of recombination (blue/total plaques) is increased by a factor of 50-80 as a function of protein concentration and of incubation time. The maximal frequency observed is 5 X 10(-5). There is no increase over the background when extracts are boiled. Electrophoresis and electron microscopy of DNA's incubated with the extracts show the formation of recombination intermediates with single strand exchange. Restriction analysis of recombined DNA confirms that the process corresponds to targeted sequence exchange. These data allow to propose three steps for homologous recombination between two duplex DNA's: i) unpairing of the two duplexes; ii) single-strand exchange and synaptic pairing; iii) resolution of the cross-junctions. The three steps correspond to those predicted by the gene conversion model of Holliday.
Full text
PDF












Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- Bianchi M., DasGupta C., Radding C. M. Synapsis and the formation of paranemic joints by E. coli RecA protein. Cell. 1983 Oct;34(3):931–939. doi: 10.1016/0092-8674(83)90550-0. [DOI] [PubMed] [Google Scholar]
- Bianchi M., Riboli B., Magni G. E. coli recA protein possesses a strand separating activity on short duplex DNAs. EMBO J. 1985 Nov;4(11):3025–3030. doi: 10.1002/j.1460-2075.1985.tb04039.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bullock P., Champoux J. J., Botchan M. Association of crossover points with topoisomerase I cleavage sites: a model for nonhomologous recombination. Science. 1985 Nov 22;230(4728):954–958. doi: 10.1126/science.2997924. [DOI] [PubMed] [Google Scholar]
- 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]
- Glazer P. M., Sarkar S. N., Chisholm G. E., Summers W. C. DNA mismatch repair detected in human cell extracts. Mol Cell Biol. 1987 Jan;7(1):218–224. doi: 10.1128/mcb.7.1.218. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hamilton A. A., Thacker J. Gene recombination in X-ray-sensitive hamster cells. Mol Cell Biol. 1987 Apr;7(4):1409–1414. doi: 10.1128/mcb.7.4.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
- Hotta Y., Tabata S., Bouchard R. A., Piñon R., Stern H. General recombination mechanisms in extracts of meiotic cells. Chromosoma. 1985;93(2):140–151. doi: 10.1007/BF00293161. [DOI] [PubMed] [Google Scholar]
- Howard-Flanders P., West S. C., Stasiak A. Role of RecA protein spiral filaments in genetic recombination. Nature. 1984 May 17;309(5965):215–219. doi: 10.1038/309215a0. [DOI] [PubMed] [Google Scholar]
- Ikeda H., Shiozaki M. Nonhomologous recombination mediated by Escherichia coli DNA gyrase: possible involvement of DNA replication. Cold Spring Harb Symp Quant Biol. 1984;49:401–409. doi: 10.1101/sqb.1984.049.01.046. [DOI] [PubMed] [Google Scholar]
- 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]
- Kmiec E. B., Holloman W. K. Synapsis promoted by Ustilago rec1 protein. Cell. 1984 Mar;36(3):593–598. doi: 10.1016/0092-8674(84)90338-6. [DOI] [PubMed] [Google Scholar]
- 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]
- Lieber M., Smith B., Szakal A., Nelson-Rees W., Todaro G. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer. 1976 Jan 15;17(1):62–70. doi: 10.1002/ijc.2910170110. [DOI] [PubMed] [Google Scholar]
- Meselson M. S., Radding C. M. A general model for genetic recombination. Proc Natl Acad Sci U S A. 1975 Jan;72(1):358–361. doi: 10.1073/pnas.72.1.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
- Roth D. B., Wilson J. H. Relative rates of homologous and nonhomologous recombination in transfected DNA. Proc Natl Acad Sci U S A. 1985 May;82(10):3355–3359. doi: 10.1073/pnas.82.10.3355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Symington L. S., Kolodner R. Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7247–7251. doi: 10.1073/pnas.82.21.7247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas K. R., Capecchi M. R. Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene. Nature. 1986 Nov 6;324(6092):34–38. doi: 10.1038/324034a0. [DOI] [PubMed] [Google Scholar]
- 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]
- West S. C., Howard-Flanders P. Duplex-duplex interactions catalyzed by RecA protein allow strand exchanges to pass double-strand breaks in DNA. Cell. 1984 Jun;37(2):683–691. doi: 10.1016/0092-8674(84)90401-x. [DOI] [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]