Biopolym. Cell. 1990; 6(2):38-45.
Study of recombination activity in mammalian cell extracts
1Akhmedov A. T., 1Namsaraev E. A., 1Zaitseva E. M., 1Zaiisev E. N., 1Lantsov V. A.
  1. B. P. Konstantinov Institute of Nuclear Physics, Academy of Sciences of the USSR
    Gatchina, Leningrad distr., USSR

Abstract

The cell-free system utilizing nuclear extracts from human mieloblasts, HeLa cells and rat testes to initiate recombination events between homologous plasmids containing different mutant alleles of the Tc gene has been described. The incubation of the plasmids with nuclear extracts increased the frequency of Tcr recombinants from 2.6·105 to 2.7·10-3. The reaction required Mg2+ and was ATP- and dNTP-independent. In addition while using a sensitive homologous recombination strand-transfer assay ATP-independent strand-transfer activity from nuclear extracts of rat testis have been partially purified. It was homology-dependent and required the presence of Mg2+. Similarities between recombinase activity and other known eukaryotic recombinases are under discussion.

References

[1] Davies KE, Robson KJ. Molecular analysis of human monogenic diseases. Bioessays. 1987;6(6):247-53.
[2] Anderson WF. Prospects for human gene therapy. Science. 1984;226(4673):401-9.
[3] Maniatis T. Targeting in mammalian cells. Nature. 1985 Sep 19-25;317(6034):205-6.
[4] Marx JL. Gene therapy--so near and yet so far away. Science. 1986;232(4752):824-5.
[5] Lantsov VA. Bacterial RecA protein: a biochemical, genetic and physicochemical analysis. Genetika. 1985;21(9):1413-27.
[6] Cox MM, Lehman IR. Enzymes of general recombination. Annu Rev Biochem. 1987;56:229-62.
[7] Kmiec E, Holloman WK. Homologous pairing of DNA molecules promoted by a protein from Ustilago. Cell. 1982;29(2):367-74.
[8] Kenne K, Ljungquist S. A DNA-recombinogenic activity in human cells. Nucleic Acids Res. 1984;12(7):3057-68.
[9] Hotta Y, Tabata S, Bouchard RA, Pi?on R, Stern H. General recombination mechanisms in extracts of meiotic cells. Chromosoma. 1985;93(2):140-51.
[10] Hsieh P, Meyn MS, Camerini-Otero RD. Partial purification and characterization of a recombinase from human cells. Cell. 1986;44(6):885-94.
[11] Cassuto E, Lightfoot LA, Howard-Flanders P. Partial purification of an activity from human cells that promotes homologous pairing and the formation of heteroduplex DNA in the presence of ATP. Mol Gen Genet. 1987;208(1-2):10-4.
[12] Ganea D, Moore P, Chekuri L, Kucherlapati R. Characterization of an ATP-dependent DNA strand transferase from human cells. Mol Cell Biol. 1987;7(9):3124-30.
[13] Kolodner R, Evans DH, Morrison PT. Purification and characterization of an activity from Saccharomyces cerevisiae that catalyzes homologous pairing and strand exchange. Proc Natl Acad Sci U S A. 1987;84(16):5560-4.
[14] Lopez B, Rousset S, Coppey J. Homologous recombination intermediates between two duplex DNA catalysed by human cell extracts. Nucleic Acids Res. 1987;15(14):5643-55.
[15] Eisen A, Camerini-Otero RD. A recombinase from Drosophila melanogaster embryos. Proc Natl Acad Sci U S A. 1988;85(20):7481-5.
[16] Fishel RA, Detmer K, Rich A. Identification of homologous pairing and strand-exchange activity from a human tumor cell line based on Z-DNA affinity chromatography. Proc Natl Acad Sci U S A. 1988;85(1):36-40.
[17] McCarthy JG, Sander M, Lowenhaupt K, Rich A. Sensitive homologous recombination strand-transfer assay: partial purification of a Drosophila melanogaster enzyme and detection of sequence effects on the strand-transfer activity of RecA protein. Proc Natl Acad Sci U S A. 1988;85(16):5854-8.
[18] Sugino A, Nitiss J, Resnick MA. ATP-independent DNA strand transfer catalyzed by protein(s) from meiotic cells of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1988;85(11):3683-7.
[19] Maniatis T., Fritsch E. F., Sambrook J. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Lab. press, 1982 545 p.
[20] DNA Cloning. First edition. Ed. D. Glover. Oxford, IRL Press, 1985; 245 p.
[21] Kalb VF Jr, Bernlohr RW. A new spectrophotometric assay for protein in cell extracts. Anal Biochem. 1977;82(2):362-71.
[22] Symington LS, Fogarty LM, Kolodner R. Genetic recombination of homologous plasmids catalyzed by cell-free extracts of Saccharomyces cerevisiae. Cell. 1983;35(3 Pt 2):805-13.
[23] Darby V, Blattner F. Homologous recombination catalyzed by mammalian cell extracts in vitro. Science. 1984;226(4679):1213-5.
[24] Kucherlapati RS, Spencer J, Moore PD. Homologous recombination catalyzed by human cell extracts. Mol Cell Biol. 1985;5(4):714-20.