Academic journal article Genetics

Smc5p Promotes Faithful Chromosome Transmission and DNA Repair in Saccharomyces Cerevisiae

Academic journal article Genetics

Smc5p Promotes Faithful Chromosome Transmission and DNA Repair in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

Heterodimers of structural maintenance of chromosomes (SMC) proteins form the core of several protein complexes involved in the organization of DNA, including condensation and cohesion of the chromosomes at metaphase. The functions of the complexes with a heterodimer of Smc5p and Smc6p are less clear. To better understand them, we created two S. cerevisiae strains bearing temperature-sensitive alleles of SMC5. When shifted to the restrictive temperature, both mutants lose viability gradually, concomitant with the appearance of nuclear abnormalities and phosphorylation of the Rad53p DNA damage checkpoint protein. Removal of Rad52p or overexpression of the SUMO ligase Mms21p partially suppresses the temperature sensitivity of smc5 strains and increases their survival at the restrictive temperature. At the permissive temperature, smc5-31 but not smc5-33 cells exhibit hypersensitivity to several DNA-damaging agents despite induction of the DNA damage checkpoint. Similarly, smc5-31 but not smc5-33 cells are killed by overexpression of the SUMO ligase-defective Mms21-SAp but not by overexpression of wild-type Mms21p. Both smc5 alleles are synthetically lethal with mms21-SA and exhibit Rad52p-independent chromosome fragmentation and loss at semipermissive temperatures. Our data indicate a critical role for the S. cerevisiae Smc5/6-containing complexes in both DNA repair and chromosome segregation.

STRUCTURAL maintenance of chromosomes (SMC) proteins hâve central roles in the organization, maintenance, and segregation of chromosomes. The single condensin complex in Saccharomyces cerevisiae and Schizosaccharomyces pombe compacts DNA, especially during cell division (STRUNNIKOV et al. 1995). In vertebrate cells, one condensin complex has the same role, while a second condensin orders the compacted DNA into chromatids (HiRANO 1998; ONO et al. 2003). During and after DNA replication and condensation, sister chromatids are linked together by the cohesin SMC complex throughout condensin-mediated compaction until the beginning of anaphase (GRUBER et al. 2003). The cell has also exploited the SMC complexes for DNA manipulation outside of mitosis. In a variety of organisms, different combinations of SMC proteins and non-SMC subunits are used to assist meiotic division, recombinational DNA repair, and dosage compensation (JESSBERGER 2002). While these extramitotic functions of SMC complexes are generally organism specific, cell division in all eukaryotic cells requires a third class of SMC complex, the Smc5/6 complex, the normal cellular role of which has recently begun to emerge.

Radiation-sensitive mutants in the S. pombe smc6 gene (also known as radlS, see Table 1) were first isolated in 1975; the wild-type gene was cloned by complementation of the smco-XUV sensitivity phenotype in 1995 (NASIM and SMITH 1975; LEHMANN et al. 1995). Cell division in the absence of wild-type Smc6 results in lethality concomitant with highly abnormal nuclear DNA morphology (LEHMANN et al. 1995; VERKADE et al. 1999; FOUSTERI and LEHMANN 2000; HARVEY et al. 2004). Smc6 mutants are unable to repair DNA damage caused by several exogenous agents and, despite normal initiation of the DNA damage checkpoint response, are unable to prevent mitosis in the presence of unrepaired damage (VERKADE et al. 1999; HARVEY et al. 2004). Various mutant alleles of smc6 have revealed that Smc6 has two roles in the cell: a nonessential role in recombinational DNA damage repair and a poorly understood essential role in maintaining genomic stability (FOUSTERI and LEHMANN 2000).

S. pombe Smc6 exists in a high-molecular-weight complex with Smc5 and several other proteins (FouSTERi and LEHMANN 2000). The other non-SMC elements (NSEs) (Nsel, Nse2, Nse3, and Rad62/Nse4) were identified by mass-spectrometric analysis of affinity-purified complexes (McDoNALD et al. 2003; MORIKAWA et al. 2004; PEBERNARD et al. 2004; SERGEANT et al. 2005). Mutants of the NSEs also exhibit nuclear fragmentation and a sensitivity to DNA damage epistatic to the homologous recombination-dependent repair pathway (McDoNALD et al. …

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