Rad62 Protein Functionally and Physically Associates with the Smc5/Smc6 Protein Complex and Is Required for Chromosome Integrity and Recombination Repair in Fission Yeast

Abstract

Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination.Mutants in Schizosaccharomyces pombe rad2, Saccharomyces cerevisiae RAD27, and Escherichia coli polA, all of which are defective in processing Okazaki fragments, are synthetically lethal with mutations in recombination repair genes (8,11,23,24,35,37,40,43,44). In these mutants, double strand breaks (DSBs) are thought to be produced when replication forks encounter the single strand gaps or nicks remaining unsealed due to the inefficient processing of Okazaki fragments, so they require a highly efficient capacity to repair DSBs for survival. In an effort to identify novel genes involved in recombination repair, we isolated mutants that were hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2⌬ and cloned the genes by complementation of the MMS sensitivity. Genes identified in this way include rhp57 (44), rad60 (35), rad32, nbs1 (45), and fdh1, which encodes an F-box DNA helicase (our unpublished data). A recent genome-wide search for mutations synthetically lethal with rad27 in S. cerevisiae identified mutations in all of the well-studied recombination repair genes including RAD52, RAD50, MRE11, XRS2 RAD51, RAD55, RAD57, RAD54, SGS1, MUS81, and MMS4 (43). The screen also identified genes involved in checkpoint regulation (RAD9, RAD17, RAD24, and DDC1) and those related to regulation of chromatin structure (CAC2, ESC2, HST1, and HST3).We report here a novel gene, rad62, which was identified by isolating mutants that were hypersensitive to MMS and synthetically lethal with rad2 mutation and cloning the gene that complemented the MMS sensitivity of such a mutant. rad62 mutants show very similar phenotypes to those of rad60 mutants (7, 35) and rad18 mutants (30, 47). They are hypersensitive to UV, MMS, and gamma rays, epistatic with rhp51 with respect to the damage sensitivity, requir...

Antagonism of Chk1 Signaling in the G2 DNA Damage Checkpoint by Dominant Alleles of Cdr1

“…Construction of cdr1-Flag3-His6 strains: A Flag 3 -His 6 tag was integrated into the 39-end of the cdr1 ORF using PCR products derived from pFA6a-Flag 3 -His 6 -kanMX6 (Morikawa et al 2004). G418-resistant colonies were selected and checked for correct integration in the genome by PCR with primers inside and outside the ORF of the cdr1 gene and by Western blotting.…”

Smc5p Promotes Faithful Chromosome Transmission and DNA Repair in Saccharomyces cerevisiae

“…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) (Nse1, Nse2, Nse3, and Rad62/Nse4) were identified by mass-spectrometric analysis of affinity-purified complexes 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 1 recombination-dependent repair pathway Morikawa et al 2004;Pebernard et al 2004).…”

Smc5-Smc6 Complex Preserves Nucleolar Integrity in S. cerevisiae

“…There are six classes of Smc proteins named Smc1 to 6 forming three distinct heterodimers. The Smc1-Smc3 heterodimer in cohesin, the Smc2-Smc4 heterodimer in condensin and the Smc5-Smc6 heterodimer which forms the core of a large protein complex [3][4][5][6][7][8][9][10][11] which specific function is still unclear and hence the complex remains functionally unnamed.…”

“…14 Besides Smc5p and Smc6p 3 the complex has four (in S.pombe) to six (in S.cerevisiae) NonSmc Elements, Nse. [4][5][6][7][8][9][10][11] NonSmc subunits of the complex have been shown to posses biochemical activities, such as Nse2, which is a SUMO E3 ligase. 11,15 Mutants in different subunits are all sensitive to a variety of DNA damaging agents.…”