Yeast Ku as a regulator of chromosomal DNA end structure

Science

Volumn 280, Issue 5364, 1998, Pages 741-744

  Gravel, Serge ^a   Larrivée, Michel ^a   Labrecque, Pascale ^a   Wellinger, Raymund J ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; CELL CYCLE S PHASE; CHROMOSOME; CROSS LINKING; DNA REPLICATION ORIGIN; GENE DUPLICATION; NONHUMAN; PRIORITY JOURNAL; TELOMERE; YEAST;

EID: 0032076127     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5364.741     Document Type: Article

References (44)

1. D. B. Roth, T. Lindahl, M. Gellert, Curr. Biol. 5, 496 (1995): D. T. Weaver, Trends Genet. 11, 388 (1995); M. R. Lieber, U. Grawunder, X. Wu, M. Yaneva, Curr. Opin. Genet. Dev. 7, 99 (1997); S. P. Jackson and P. A. Jeggo, Trends Biochem. Sci. 20, 412 (1995).
2. D. B. Roth, T. Lindahl, M. Gellert, Curr. Biol. 5, 496 (1995): D. T. Weaver, Trends Genet. 11, 388 (1995); M. R. Lieber, U. Grawunder, X. Wu, M. Yaneva, Curr. Opin. Genet. Dev. 7, 99 (1997); S. P. Jackson and P. A. Jeggo, Trends Biochem. Sci. 20, 412 (1995).
3. D. B. Roth, T. Lindahl, M. Gellert, Curr. Biol. 5, 496 (1995): D. T. Weaver, Trends Genet. 11, 388 (1995); M. R. Lieber, U. Grawunder, X. Wu, M. Yaneva, Curr. Opin. Genet. Dev. 7, 99 (1997); S. P. Jackson and P. A. Jeggo, Trends Biochem. Sci. 20, 412 (1995).
4. D. B. Roth, T. Lindahl, M. Gellert, Curr. Biol. 5, 496 (1995): D. T. Weaver, Trends Genet. 11, 388 (1995); M. R. Lieber, U. Grawunder, X. Wu, M. Yaneva, Curr. Opin. Genet. Dev. 7, 99 (1997); S. P. Jackson and P. A. Jeggo, Trends Biochem. Sci. 20, 412 (1995).
5. T. Mimori and J. A. Hardin, J. Biol. Chem. 261, 10375 (1986); S. Paillard and F. Strauss, Nucleic Acids Res. 19, 5619 (1991).
6. T. Mimori and J. A. Hardin, J. Biol. Chem. 261, 10375 (1986); S. Paillard and F. Strauss, Nucleic Acids Res. 19, 5619 (1991).
7. H. Feldmann and E.-L. Winnacker, J. Biol. Chem. 268, 12895 (1993); H. Feldmann et al., ibid. 271, 27765 (1996).
8. H. Feldmann and E.-L. Winnacker, J. Biol. Chem. 268, 12895 (1993); H. Feldmann et al., ibid. 271, 27765 (1996).
9. S. J. Boulton and S. P. Jackson, Nucleic Acids Res. 24, 4639 (1996).
10. G. T. Milne, S. Jin, K. B. Shannon, D. T. Weaver. Mol. Cell. Biol. 16, 4189 (1996).
11. G. J. Mages, H. M. Feldmann, E.-L. Winnacker, J. Biol. Chem. 271, 7910 (1996); W. Siede, A. A. Friedl, I. Dianova, F. Eckardt-Schupp, E. C. Friedberg, Genetics 142, 91 (1996); Y. Tsukamoto, J. Kato, H. Ikeda, Nucleic Acids Res. 24, 2067 (1996); S. J. Boulton and S. P. Jackson, EMBO J. 15, 5093 (1996).
12. G. J. Mages, H. M. Feldmann, E.-L. Winnacker, J. Biol. Chem. 271, 7910 (1996); W. Siede, A. A. Friedl, I. Dianova, F. Eckardt-Schupp, E. C. Friedberg, Genetics 142, 91 (1996); Y. Tsukamoto, J. Kato, H. Ikeda, Nucleic Acids Res. 24, 2067 (1996); S. J. Boulton and S. P. Jackson, EMBO J. 15, 5093 (1996).
13. G. J. Mages, H. M. Feldmann, E.-L. Winnacker, J. Biol. Chem. 271, 7910 (1996); W. Siede, A. A. Friedl, I. Dianova, F. Eckardt-Schupp, E. C. Friedberg, Genetics 142, 91 (1996); Y. Tsukamoto, J. Kato, H. Ikeda, Nucleic Acids Res. 24, 2067 (1996); S. J. Boulton and S. P. Jackson, EMBO J. 15, 5093 (1996).
14. G. J. Mages, H. M. Feldmann, E.-L. Winnacker, J. Biol. Chem. 271, 7910 (1996); W. Siede, A. A. Friedl, I. Dianova, F. Eckardt-Schupp, E. C. Friedberg, Genetics 142, 91 (1996); Y. Tsukamoto, J. Kato, H. Ikeda, Nucleic Acids Res. 24, 2067 (1996); S. J. Boulton and S. P. Jackson, EMBO J. 15, 5093 (1996).
15. S. E. Porter, P. W. Greenwell, K. B. Ritchie, T. D. Petes, Nucleic Acids Res. 24, 582 (1996).
16. Y. Tsukamoto, J. Kato, H. Ikeda, Nature 388, 900 (1997).
17. J. Shampay, J. W. Szostak, E. H. Blackburn, ibid. 310, 154 (1984).
18. E. H. Blackburn and C. W. Greider, Eds., Telomeres (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ed. 1, 1995); C. W. Greider, Annu. Rev. Biochem. 65, 337 (1996); V. A. Zakian, Annu. Rev. Genet. 30, 141 (1996); R. J. Wellinger and D. Sen, Eur. J. Cancer 33, 735 (1997).
19. E. H. Blackburn and C. W. Greider, Eds., Telomeres (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ed. 1, 1995); C. W. Greider, Annu. Rev. Biochem. 65, 337 (1996); V. A. Zakian, Annu. Rev. Genet. 30, 141 (1996); R. J. Wellinger and D. Sen, Eur. J. Cancer 33, 735 (1997).
20. E. H. Blackburn and C. W. Greider, Eds., Telomeres (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ed. 1, 1995); C. W. Greider, Annu. Rev. Biochem. 65, 337 (1996); V. A. Zakian, Annu. Rev. Genet. 30, 141 (1996); R. J. Wellinger and D. Sen, Eur. J. Cancer 33, 735 (1997).
21. E. H. Blackburn and C. W. Greider, Eds., Telomeres (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ed. 1, 1995); C. W. Greider, Annu. Rev. Biochem. 65, 337 (1996); V. A. Zakian, Annu. Rev. Genet. 30, 141 (1996); R. J. Wellinger and D. Sen, Eur. J. Cancer 33, 735 (1997).
22. R. J. Wellinger, A. J. Wolf, V. A. Zakian, Cell 72, 51 (1993).
23. I. Dionne and R. J. Wellinger, Proc. Natl. Acad. Sci. U.S.A. 93, 13902 (1996).
24. R. J. Wellinger, K. Ethier, P. Labrecque, V. A. Zakian, Cell 85, 423 (1996).
25. The Hartwell collection of ts yeast strains (28) was analyzed for DNA content, and strains that arrested with a 2n content were selected for further study. Strains H742 consistently yielded strong telomeric signals in native gels, indicative of abnormally long G-strand extensions. After three successive backcrosses, the ts phenotype associated with strain H742 segregated 2:2 in 13 of 14 tetrads analyzed. Four of these tetrads were analyzed for telomere length and DNA end structure. In all four short telomeres and an overhang of the G-rich strand cosegregated with the ts phenotype. As for wild-type strains, ss C strands were never detectable (23). The original designation of the mutation in these strains was get1-1 (G-strand extensions at telomeres), but once it was realized that GET1 was allelic to YKU80/ HDF2 (see below), this designation was changed to conform to the currently used gene nomenclature. The precise nature of the get1-1 mutation is unknown, but because strains harboring this mutation and strains harboring a deletion of the YKU80 gene display identical phenotypes in all assays used here, we assume that the get1-1 allele produces a non-functional Yku80p. The following individual segregants after the outcrossing were used: RWY738d [MATa, (GET1)YKU80], RWY737d and RWY738a [MATa, (get1-1)yku80], and RWY739b [MATα, (get1-1)yku80].
26. Strain RWY737d or RWY739b (14) was crossed to strains DWY290, DWY291, DWY292, and DWY293 (29) and the indicated phenotypes were tested in diploids (23).
27. The YKU80 gene encoding a COOH-terminal Myc tag was obtained from genomic DNA by polymerase chain reactions (PCRs). This gene contained nucleotides -638 to +1885 with respect to the first ATG of the genomic YKU80 sequence and 33 nucleotides encoding the 10 amino acids for the Myc tag plus a stop codon at the 3′ end. The protein thus was a full-length, wild-type Yku80p fused to the Myc epitope recognized by the 9E10 monoclonal antibody (Boehringer Mannheim). A 2.5-kb Hind III-Not I fragment containing this gene was then subcloned into the yeast shuttle vector pRS316 (30), yielding pKU80-myc. This plasmid, when introduced into strain RWY737d [(get1-1)yku80], fully complemented the ts, telomeric length, and DNA end-structure phenotypes (23) and was also used in the immunoprecipitation studies (Fig. 3). A similar construct has been shown to complement DNA repair deficiencies in yku80Δ strains and the tagged protein was also fully functional in end-binding assays with yeast extracts (5).
28. I. R. Lehmann and A. L. Nussbaum, J. Biol. Chem. 239, 2628 (1964).
29. The length was estimated from experiments with a variety of restriction enzymes and longer gels (S. Gravel, M. Larrivée, R. J. Wellinger, unpublished data).
30. V. A. Zakian, Cell 82, 685 (1995).
31. A. J. Lustig and T. D. Petes, Proc. Natl. Acad. Sci. U.S.A. 83, 1398 (1986).
32. G. Barnes and D. Rio, ibid. 94, 867 (1997).
33. S. Strahl-Bolsinger, A. Hecht, K. Luo, M. Grunstein, Genes Dev. 11, 83 (1997); V. Orlando and R. Paro, Cell 75, 1187 (1993).
34. S. Strahl-Bolsinger, A. Hecht, K. Luo, M. Grunstein, Genes Dev. 11, 83 (1997); V. Orlando and R. Paro, Cell 75, 1187 (1993).
35. S. Gravel, M. Larrivée, R. J. Wellinger, unpublished data.
36. The competitor DNA was a pVZ1 plasmid containing about 280 bp of telomeric repeats derived from plasmid pYLPV (11). This plasmid was digested with Not I to yield a linear DNA fragment of 3.5 kb that had a block of 280 bp of telomeric repeats close to one of the ends. pVZ1 is a 3.2-kb bacterial vector derived from pBS + (Stratagene).
37. - cells (23).
38. M.S. Singer and D. E. Gottschling, Science 266, 404 (1994).
39. C. I. Nugent, T. R. Hughes, N. F. Lue, V. Lundblad, ibid. 274, 249 (1996).
40. L. H. Hartwell, J. Bacteriol. 93, 1662 (1967).
41. Strains DWY290 (hdf1Δ::TRP1, YKU80), DWY291 (HDF1, YKU80), DWY292 (HDF1, yku80Δ::URA3), and DWY293 (hdf1Δ::TRP1, yku80Δ::URA3) were from D. Weaver (5). Strain KWRY100 (MATa, tel1Δ:: HIS3) harbors a complete deletion of the TEL1 coding sequence and was from K. Runge.
42. R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989).
43. est mutation (27) or with a tlc1Δ strain (26).
44. We thank E. A. Siewert and M. Winey for analyzing and providing us with the ts mutant strains; D. Weaver, K. Runge, V. Lundblad, and D. Gottschling for supplying yeast strains; K. Runge for insightful suggestions; B. Chabot and K. Runge for input on the manuscript; and V. Lundblad for sharing unpublished data. Supported by grants from the Canadian MRC (MT-12616) and the National Cancer Institute of Canada (007103). R.J.W. is a Chercheur-Boursier Senior of the FRSQ.