Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells (original) (raw)

Abstract

Repair of DNA damage resulting in double-strand breaks (DSBs) is controlled by gene products executing homologous recombination or end-joining pathways. The MRE11 gene has previously been implicated in DSB repair in the yeast Saccharomyces cerevisiae . Here we have developed a methodology to study the roles of the murine Mre11 homolog in pluripotent embryonic stem cells. Using a gene targeting approach, a triple LoxP site cassette was inserted into a region of MRE11 genomic DNA flanking conserved phosphodiesterase motifs. The addition of Cre recombinase activity promotes deletions of three types that can be scored. We find that deletion at phosphodiesterase motif III encoded in the N-terminus of Mre11 is acheived in the presence of a wild-type MRE11 allele. However, when the wild-type MRE11 allele is inactivated by gene targeted insertion of a neo marker, only Cre recombination events that allow expression of wild-type Mre11 protein are observed. Therefore, Mre11 is required for normal cell proliferation. This methodology introduces a means to study important regions of essential genes in cell culture models.

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Selected References

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  1. Ajimura M., Leem S. H., Ogawa H. Identification of new genes required for meiotic recombination in Saccharomyces cerevisiae. Genetics. 1993 Jan;133(1):51–66. doi: 10.1093/genetics/133.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alani E., Cao L., Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987 Aug;116(4):541–545. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bezzubova O., Silbergleit A., Yamaguchi-Iwai Y., Takeda S., Buerstedde J. M. Reduced X-ray resistance and homologous recombination frequencies in a RAD54-/- mutant of the chicken DT40 cell line. Cell. 1997 Apr 18;89(2):185–193. doi: 10.1016/s0092-8674(00)80198-1. [DOI] [PubMed] [Google Scholar]
  4. Connelly J. C., Leach D. R. The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination. Genes Cells. 1996 Mar;1(3):285–291. doi: 10.1046/j.1365-2443.1996.23024.x. [DOI] [PubMed] [Google Scholar]
  5. Dolganov G. M., Maser R. S., Novikov A., Tosto L., Chong S., Bressan D. A., Petrini J. H. Human Rad50 is physically associated with human Mre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair. Mol Cell Biol. 1996 Sep;16(9):4832–4841. doi: 10.1128/mcb.16.9.4832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Essers J., Hendriks R. W., Swagemakers S. M., Troelstra C., de Wit J., Bootsma D., Hoeijmakers J. H., Kanaar R. Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination. Cell. 1997 Apr 18;89(2):195–204. doi: 10.1016/s0092-8674(00)80199-3. [DOI] [PubMed] [Google Scholar]
  7. Game J. C., Mortimer R. K. A genetic study of x-ray sensitive mutants in yeast. Mutat Res. 1974 Sep;24(3):281–292. doi: 10.1016/0027-5107(74)90176-6. [DOI] [PubMed] [Google Scholar]
  8. Gu H., Marth J. D., Orban P. C., Mossmann H., Rajewsky K. Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science. 1994 Jul 1;265(5168):103–106. doi: 10.1126/science.8016642. [DOI] [PubMed] [Google Scholar]
  9. Gu H., Zou Y. R., Rajewsky K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell. 1993 Jun 18;73(6):1155–1164. doi: 10.1016/0092-8674(93)90644-6. [DOI] [PubMed] [Google Scholar]
  10. Hakem R., de la Pompa J. L., Sirard C., Mo R., Woo M., Hakem A., Wakeham A., Potter J., Reitmair A., Billia F. The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse. Cell. 1996 Jun 28;85(7):1009–1023. doi: 10.1016/s0092-8674(00)81302-1. [DOI] [PubMed] [Google Scholar]
  11. Ivanov E. L., Korolev V. G., Fabre F. XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination. Genetics. 1992 Nov;132(3):651–664. doi: 10.1093/genetics/132.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jin S., Inoue S., Weaver D. T. Functions of the DNA dependent protein kinase. Cancer Surv. 1997;29:221–261. [PubMed] [Google Scholar]
  13. Johzuka K., Ogawa H. Interaction of Mre11 and Rad50: two proteins required for DNA repair and meiosis-specific double-strand break formation in Saccharomyces cerevisiae. Genetics. 1995 Apr;139(4):1521–1532. doi: 10.1093/genetics/139.4.1521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kramer K. M., Brock J. A., Bloom K., Moore J. K., Haber J. E. Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events. Mol Cell Biol. 1994 Feb;14(2):1293–1301. doi: 10.1128/mcb.14.2.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Leach D. R., Lloyd R. G., Coulson A. F. The SbcCD protein of Escherichia coli is related to two putative nucleases in the UvrA superfamily of nucleotide-binding proteins. Genetica. 1992;87(2):95–100. doi: 10.1007/BF00120998. [DOI] [PubMed] [Google Scholar]
  16. Leach D. R. Long DNA palindromes, cruciform structures, genetic instability and secondary structure repair. Bioessays. 1994 Dec;16(12):893–900. doi: 10.1002/bies.950161207. [DOI] [PubMed] [Google Scholar]
  17. Milne G. T., Jin S., Shannon K. B., Weaver D. T. Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Aug;16(8):4189–4198. doi: 10.1128/mcb.16.8.4189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Moore J. K., Haber J. E. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae. Mol Cell Biol. 1996 May;16(5):2164–2173. doi: 10.1128/mcb.16.5.2164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mortensen R. M., Conner D. A., Chao S., Geisterfer-Lowrance A. A., Seidman J. G. Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol. 1992 May;12(5):2391–2395. doi: 10.1128/mcb.12.5.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nussenzweig A., Chen C., da Costa Soares V., Sanchez M., Sokol K., Nussenzweig M. C., Li G. C. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature. 1996 Aug 8;382(6591):551–555. doi: 10.1038/382551a0. [DOI] [PubMed] [Google Scholar]
  21. Petrini J. H., Walsh M. E., DiMare C., Chen X. N., Korenberg J. R., Weaver D. T. Isolation and characterization of the human MRE11 homologue. Genomics. 1995 Sep 1;29(1):80–86. doi: 10.1006/geno.1995.1217. [DOI] [PubMed] [Google Scholar]
  22. Petrini J. H., Xiao Y., Weaver D. T. DNA ligase I mediates essential functions in mammalian cells. Mol Cell Biol. 1995 Aug;15(8):4303–4308. doi: 10.1128/mcb.15.8.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Scully R., Chen J., Plug A., Xiao Y., Weaver D., Feunteun J., Ashley T., Livingston D. M. Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell. 1997 Jan 24;88(2):265–275. doi: 10.1016/s0092-8674(00)81847-4. [DOI] [PubMed] [Google Scholar]
  24. Sharples G. J., Leach D. R. Structural and functional similarities between the SbcCD proteins of Escherichia coli and the RAD50 and MRE11 (RAD32) recombination and repair proteins of yeast. Mol Microbiol. 1995 Sep;17(6):1215–1217. doi: 10.1111/j.1365-2958.1995.mmi_17061215_1.x. [DOI] [PubMed] [Google Scholar]
  25. Tavassoli M., Shayeghi M., Nasim A., Watts F. Z. Cloning and characterisation of the Schizosaccharomyces pombe rad32 gene: a gene required for repair of double strand breaks and recombination. Nucleic Acids Res. 1995 Feb 11;23(3):383–388. doi: 10.1093/nar/23.3.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Tsuzuki T., Fujii Y., Sakumi K., Tominaga Y., Nakao K., Sekiguchi M., Matsushiro A., Yoshimura Y., MoritaT Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6236–6240. doi: 10.1073/pnas.93.13.6236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weaver D. T. What to do at an end: DNA double-strand-break repair. Trends Genet. 1995 Oct;11(10):388–392. doi: 10.1016/s0168-9525(00)89121-0. [DOI] [PubMed] [Google Scholar]
  29. Zhu C., Bogue M. A., Lim D. S., Hasty P., Roth D. B. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell. 1996 Aug 9;86(3):379–389. doi: 10.1016/s0092-8674(00)80111-7. [DOI] [PubMed] [Google Scholar]