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. 1998 Oct;150(2):591–600. doi: 10.1093/genetics/150.2.591

Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11.

D A Bressan 1, H A Olivares 1, B E Nelms 1, J H Petrini 1
PMCID: PMC1460356  PMID: 9755192

Abstract

Saccharomyces cerevisiae Mre11, Rad50, and Xrs2 function in a protein complex that is important for nonhomologous recombination. Null mutants of MRE11, RAD50, and XRS2 are characterized by ionizing radiation sensitivity and mitotic interhomologue hyperrecombination. We mutagenized the four highly conserved phosphoesterase signature motifs of Mre11 to create mre11-11, mre11-2, mre11-3, and mre11-4 and assessed the functional consequences of these mutant alleles with respect to mitotic interhomologue recombination, chromosome loss, ionizing radiation sensitivity, double-strand break repair, and protein interaction. We found that mre11 mutants that behaved as the null were sensitive to ionizing radiation and deficient in double-strand break repair. We also observed that these null mutants exhibited a hyperrecombination phenotype in mitotic cells, consistent with previous reports, but did not exhibit an increased frequency of chromosome loss. Differential ionizing radiation sensitivities among the hypomorphic mre11 alleles correlated with the trends observed in the other phenotypes examined. Two-hybrid interaction testing showed that all but one of the mre11 mutations disrupted the Mre11-Rad50 interaction. Mutagenesis of the phosphoesterase signatures in Mre11 thus demonstrated the importance of these conserved motifs for recombinational DNA repair.

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Selected References

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

  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. Alani E., Padmore R., Kleckner N. Analysis of wild-type and rad50 mutants of yeast suggests an intimate relationship between meiotic chromosome synapsis and recombination. Cell. 1990 May 4;61(3):419–436. doi: 10.1016/0092-8674(90)90524-i. [DOI] [PubMed] [Google Scholar]
  4. Bai Y., Symington L. S. A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 1996 Aug 15;10(16):2025–2037. doi: 10.1101/gad.10.16.2025. [DOI] [PubMed] [Google Scholar]
  5. Barnes G., Rio D. DNA double-strand-break sensitivity, DNA replication, and cell cycle arrest phenotypes of Ku-deficient Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):867–872. doi: 10.1073/pnas.94.3.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Becker D. M., Fikes J. D., Guarente L. A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1968–1972. doi: 10.1073/pnas.88.5.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carney J. P., Maser R. S., Olivares H., Davis E. M., Le Beau M., Yates J. R., 3rd, Hays L., Morgan W. F., Petrini J. H. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell. 1998 May 1;93(3):477–486. doi: 10.1016/s0092-8674(00)81175-7. [DOI] [PubMed] [Google Scholar]
  8. Cohen P. T., Collins J. F., Coulson A. F., Berndt N., da Cruz e Silva O. B. Segments of bacteriophage lambda (orf 221) and phi 80 are homologous to genes coding for mammalian protein phosphatases. Gene. 1988 Sep 15;69(1):131–134. doi: 10.1016/0378-1119(88)90385-x. [DOI] [PubMed] [Google Scholar]
  9. Connelly C., Hieter P. Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression. Cell. 1996 Jul 26;86(2):275–285. doi: 10.1016/S0092-8674(00)80099-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Connelly J. C., de Leau E. S., Okely E. A., Leach D. R. Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli. J Biol Chem. 1997 Aug 8;272(32):19819–19826. doi: 10.1074/jbc.272.32.19819. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Estojak J., Brent R., Golemis E. A. Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol. 1995 Oct;15(10):5820–5829. doi: 10.1128/mcb.15.10.5820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Game J. C. DNA double-strand breaks and the RAD50-RAD57 genes in Saccharomyces. Semin Cancer Biol. 1993 Apr;4(2):73–83. [PubMed] [Google Scholar]
  15. 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]
  16. Gerring S. L., Spencer F., Hieter P. The CHL 1 (CTF 1) gene product of Saccharomyces cerevisiae is important for chromosome transmission and normal cell cycle progression in G2/M. EMBO J. 1990 Dec;9(13):4347–4358. doi: 10.1002/j.1460-2075.1990.tb07884.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Griffith J. P., Kim J. L., Kim E. E., Sintchak M. D., Thomson J. A., Fitzgibbon M. J., Fleming M. A., Caron P. R., Hsiao K., Navia M. A. X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell. 1995 Aug 11;82(3):507–522. doi: 10.1016/0092-8674(95)90439-5. [DOI] [PubMed] [Google Scholar]
  18. Hartwell L. H., Smith D. Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. Genetics. 1985 Jul;110(3):381–395. doi: 10.1093/genetics/110.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hieter P., Mann C., Snyder M., Davis R. W. Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss. Cell. 1985 Feb;40(2):381–392. doi: 10.1016/0092-8674(85)90152-7. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Ivanov E. L., Sugawara N., Fishman-Lobell J., Haber J. E. Genetic requirements for the single-strand annealing pathway of double-strand break repair in Saccharomyces cerevisiae. Genetics. 1996 Mar;142(3):693–704. doi: 10.1093/genetics/142.3.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ivanov E. L., Sugawara N., White C. I., Fabre F., Haber J. E. Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae. Mol Cell Biol. 1994 May;14(5):3414–3425. doi: 10.1128/mcb.14.5.3414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Jong A. Y., Wang B., Zhang S. Q. Pulsed field gel electrophoresis labeling method to study the pattern of Saccharomyces cerevisiae chromosomal DNA synthesis during the G1/S phase of the cell cycle. Anal Biochem. 1995 May 1;227(1):32–39. doi: 10.1006/abio.1995.1249. [DOI] [PubMed] [Google Scholar]
  25. Kuzminov A. Collapse and repair of replication forks in Escherichia coli. Mol Microbiol. 1995 May;16(3):373–384. doi: 10.1111/j.1365-2958.1995.tb02403.x. [DOI] [PubMed] [Google Scholar]
  26. Malone R. E., Bullard S., Hermiston M., Rieger R., Cool M., Galbraith A. Isolation of mutants defective in early steps of meiotic recombination in the yeast Saccharomyces cerevisiae. Genetics. 1991 May;128(1):79–88. doi: 10.1093/genetics/128.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Malone R. E., Ward T., Lin S., Waring J. The RAD50 gene, a member of the double strand break repair epistasis group, is not required for spontaneous mitotic recombination in yeast. Curr Genet. 1990 Aug;18(2):111–116. doi: 10.1007/BF00312598. [DOI] [PubMed] [Google Scholar]
  28. Meeks-Wagner D., Hartwell L. H. Normal stoichiometry of histone dimer sets is necessary for high fidelity of mitotic chromosome transmission. Cell. 1986 Jan 17;44(1):43–52. doi: 10.1016/0092-8674(86)90483-6. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Morrow D. M., Tagle D. A., Shiloh Y., Collins F. S., Hieter P. TEL1, an S. cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is functionally related to the yeast checkpoint gene MEC1. Cell. 1995 Sep 8;82(5):831–840. doi: 10.1016/0092-8674(95)90480-8. [DOI] [PubMed] [Google Scholar]
  31. Nairz K., Klein F. mre11S--a yeast mutation that blocks double-strand-break processing and permits nonhomologous synapsis in meiosis. Genes Dev. 1997 Sep 1;11(17):2272–2290. doi: 10.1101/gad.11.17.2272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Naom I. S., Morton S. J., Leach D. R., Lloyd R. G. Molecular organization of sbcC, a gene that affects genetic recombination and the viability of DNA palindromes in Escherichia coli K-12. Nucleic Acids Res. 1989 Oct 25;17(20):8033–8045. doi: 10.1093/nar/17.20.8033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nasmyth K. A. Temperature-sensitive lethal mutants in the structural gene for DNA ligase in the yeast Schizosaccharomyces pombe. Cell. 1977 Dec;12(4):1109–1120. doi: 10.1016/0092-8674(77)90173-8. [DOI] [PubMed] [Google Scholar]
  34. Petrini J. H., Bressan D. A., Yao M. S. The RAD52 epistasis group in mammalian double strand break repair. Semin Immunol. 1997 Jun;9(3):181–188. doi: 10.1006/smim.1997.0067. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  37. Schiestl R. H., Zhu J., Petes T. D. Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1994 Jul;14(7):4493–4500. doi: 10.1128/mcb.14.7.4493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schneider B. L., Seufert W., Steiner B., Yang Q. H., Futcher A. B. Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast. 1995 Oct;11(13):1265–1274. doi: 10.1002/yea.320111306. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sonoda E., Sasaki M. S., Buerstedde J. M., Bezzubova O., Shinohara A., Ogawa H., Takata M., Yamaguchi-Iwai Y., Takeda S. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J. 1998 Jan 15;17(2):598–608. doi: 10.1093/emboj/17.2.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Spencer F., Gerring S. L., Connelly C., Hieter P. Mitotic chromosome transmission fidelity mutants in Saccharomyces cerevisiae. Genetics. 1990 Feb;124(2):237–249. doi: 10.1093/genetics/124.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Tsubouchi H., Ogawa H. A novel mre11 mutation impairs processing of double-strand breaks of DNA during both mitosis and meiosis. Mol Cell Biol. 1998 Jan;18(1):260–268. doi: 10.1128/mcb.18.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tsukamoto Y., Kato J., Ikeda H. Effects of mutations of RAD50, RAD51, RAD52, and related genes on illegitimate recombination in Saccharomyces cerevisiae. Genetics. 1996 Feb;142(2):383–391. doi: 10.1093/genetics/142.2.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  47. Xiao Y., Weaver D. T. Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells. Nucleic Acids Res. 1997 Aug 1;25(15):2985–2991. doi: 10.1093/nar/25.15.2985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhuo S., Clemens J. C., Stone R. L., Dixon J. E. Mutational analysis of a Ser/Thr phosphatase. Identification of residues important in phosphoesterase substrate binding and catalysis. J Biol Chem. 1994 Oct 21;269(42):26234–26238. [PubMed] [Google Scholar]

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