Sir-Ku-itous Routes to Make Ends Meet

Recent results have raised the possibility that the effect of Sir proteins on NHEJ is largely a reflection of their role in regulating mating-type gene expression (1xAstrom, S.U, Okamura, S.M, and Rine, J. Nature. 1999; 397: 310Crossref | PubMed | Scopus (88)See all References, 13xLee, S.E, Pâques, F, Sylvan, J, and Haber, J.E. Curr. Biol. 1999; in pressSee all References). When SIR genes are deleted, haploid yeast cells express the normally silent mating-type cassettes, HMLα and HMRa, causing the haploid cell to have the nonmating phenotype of a MATa/MATα diploid. Cells expressing both MATa and MATα genes turn off a set of haploid-specific genes and turn on other diploid-specific genes. Thus, MATa/MATα diploids are more X-ray resistant than diploids expressing a single mating type and have higher levels of both spontaneous and DSB-induced recombination (7xHeude, M and Fabre, F. Genetics. 1993; 133: 489–498PubMedSee all References, 13xLee, S.E, Pâques, F, Sylvan, J, and Haber, J.E. Curr. Biol. 1999; in pressSee all References). Yeast cells apparently “know” they are diploid or haploid by their mating type: in nature, haploids mate and diploids don’t.It makes sense that haploid cells would wish to upregulate NHEJ, since in the G1 stage of the cell cycle they must rely on NHEJ to repair breaks, whereas diploid G1 cells always have a homologous chromosome present to act as a template for homologous recombination. Two observations support the idea that sir mutants predominantly affect NHEJ by affecting mating type (1xAstrom, S.U, Okamura, S.M, and Rine, J. Nature. 1999; 397: 310Crossref | PubMed | Scopus (88)See all References, 13xLee, S.E, Pâques, F, Sylvan, J, and Haber, J.E. Curr. Biol. 1999; in pressSee all References). First, haploid sir2 or sir3 or sir4 MATa cells that lack HMLα, and thus remain a mating, are as efficient in NHEJ as Sir+ strains. Similarly, a MATa/MATα diploid exhibits reduced end joining, compared to a diploid expressing only MATα.However, other data argue that Sir proteins actually do play at least a small role in DNA repair, even in MATa/MATα cells. Sir− MATa/MATα cells are more defective in DNA end joining and are more sensitive to MMS than Sir+ strains (13xLee, S.E, Pâques, F, Sylvan, J, and Haber, J.E. Curr. Biol. 1999; in pressSee all References, 16xMartin, S, Laroche, T, Suka, N, Grunstein, M, and Gasser, S.M. Cell. 1999; 97: 621–624Abstract | Full Text | Full Text PDF | PubMed | Scopus (364)See all References, 17xMills, K, Sinclair, D, and Guarente, L. Cell. 1999; 97: 609–612Abstract | Full Text | Full Text PDF | PubMed | Scopus (251)See all References). The importance of Sir proteins is still not fully resolved, as there are significant differences among the results from several labs, some of which could reflect strain differences or the nature of the DNA breaks. Thus, NHEJ of a single HO-induced DSB on a chromosome is reduced no more than 2-fold in a sir mutant, whereas it is reduced 200-fold in a yku70 mutant (Lee et al. 1999xLee, S.E, Pâques, F, Sylvan, J, and Haber, J.E. Curr. Biol. 1999; in pressSee all ReferencesLee et al. 1999). In contrast, Mills et al. 1999xMills, K, Sinclair, D, and Guarente, L. Cell. 1999; 97: 609–612Abstract | Full Text | Full Text PDF | PubMed | Scopus (251)See all ReferencesMills et al. 1999 show that a sir2 strain—independent of the cell’s mating type—is as sensitive to EcoRI cleavage as one deleted for YKU70.In general the evidence does not support the idea that Sir proteins could make regions near the DSB heterochromatic, possibly inhibiting 5′ to 3′ degradation of DNA by exonucleases and facilitating end joining (8xJackson, S.P. Nature. 1997; 388: 829–830Crossref | PubMed | Scopus (16)See all References, 19xTsukamoto, Y, Kato, J, and Ikeda, H. Nature. 1997; 388: 900–903Crossref | PubMed | Scopus (282)See all References). The absence of yKu70 does cause a 2-fold increase in exonuclease resection of DSB ends (Lee et al., 1998); sir mutants haven’t been tested. But there is no evidence that more extensive resection poses a problem for yeast to complete NHEJ, since many repair events are deletions that remove thousands of base pairs.Martin et al. 1999xMartin, S, Laroche, T, Suka, N, Grunstein, M, and Gasser, S.M. Cell. 1999; 97: 621–624Abstract | Full Text | Full Text PDF | PubMed | Scopus (364)See all ReferencesMartin et al. 1999 offer an alternative explanation for the movement of Sir proteins: the Sir proteins may be dispatched to sites of newly assembled chromatin, just as the chromatin assembly factor CAF1 is recruited to sites of UV damage in mammalian cells. Sir proteins would then be involved in reestablishing chromatin structure at sites of DNA damage. UV damage doesn’t cause a change in Sir protein localization (Mills et al. 1999xMills, K, Sinclair, D, and Guarente, L. Cell. 1999; 97: 609–612Abstract | Full Text | Full Text PDF | PubMed | Scopus (251)See all ReferencesMills et al. 1999), but the idea merits further study.Finally, there is the possiblity that there may be a few other genes besides HML and HMR and those near telomeres that could be subject to Sir repression. Martin et al. 1999xMartin, S, Laroche, T, Suka, N, Grunstein, M, and Gasser, S.M. Cell. 1999; 97: 621–624Abstract | Full Text | Full Text PDF | PubMed | Scopus (364)See all ReferencesMartin et al. 1999 report that an internally located ADE2 gene with an adjacent silencer becomes more repressed after DNA damage. This raises the possibility that there are normal genes that could be repressed by Sir proteins in response to DNA damage—a kind of DNA damage stress response. Microarray technology is now available to ask whether there are other SIR-regulated genes. We should also remember that telomeres and subtelomeric regions are not the only “reservoir” for these proteins. Sir2p has an incredibly complicated second job inside the nucleolus, interacting with the RENT (re pression of n ucleolar silencing and t elophase) complex, as reviewed by Garcia and Pillus 1999xGarcia, S.N and Pillus, L. Cell. 1999; 97: 825–828Abstract | Full Text | Full Text PDF | PubMed | Scopus (59)See all ReferencesGarcia and Pillus 1999 in this issue of Cell.There are many interesting findings—and an equal number of new questions—raised by these papers. Who would have thought that Ku proteins would be so critical in maintaining Sir proteins at telomeres? Why should DNA damage be acted upon or sensed differently in S phase from either G1 or G2? What is the state of EcoRI-cut DNA and its associated chromatin that allows a Ku+ cell to repair all the breaks without making dozens of deletions? What is the relationship between mating type, ploidy, and the importance of SIR genes in NHEJ?And, finally, why should the same proteins that are critical for telomere maintenance—to keep the natural ends of chromosomes from being degraded or fusing or recombining—also be needed to facilitate the joining of other broken DNA ends? Assuming that this is not simply a cosmic joke, we will have to look even more deeply into the processes of DNA repair and chromatin structures to find the answer.