Index References

The SMC proteins were first identified by genetic analysis of mutants defective in faithful chromosome segregation in budding yeast (Strunnikov et al., 1993). These proteins commonly have two coiled-coil domains linked by a hinge region, a highly conserved head domain in their amino-terminus, and a tail domain in their carboxyl-terminus (Losada and Hirano, 2005). During cell cycle progression, chromosomes undergo structural changes that are crucial for the maintenance of genomic integrity and the faithful transmission of genetic material. Among the SMC family proteins, Smc1-Smc3 and Smc2-Smc4 complexes play important roles in sister chromatid cohesion and mitotic chromosome condensation, respectively (Losada and Hirano, 2005). The function of Smc6, a component of the Smc5-Smc6 complex, has only recently begun to be addressed in detail.

SMC6 was originally isolated as the rad18⁺ mutant of Schizosaccharomyces pombe, which undergoes aberrant mitosis following exposure to genotoxic agents (Lehman et al., 1995). Smc6 has also been linked to DNA repair through the homologous recombination pathway (Lehman et al., 1995). In the budding yeast Saccharomyces cerevisiae, SMC6 and SMC5 are essential for cell viability, but some smc5 and smc6 temperature sensitive alleles have been identified and characterized (Onoda et al., 2004; Torres-Rosell et al., 2005; Cost and Cozzarelli, 2006). Previously, using a temperature-sensitive mutant strain, smc6-56, we found that Smc6 functions in the Rad52-dependent recombination repair pathway. Moreover, Smc6 is required for homologous recombination induced by exposure to methyl methanesulfonate (MMS) (Onoda et al., 2004). Taking into account the structure of Smc6, it seems likely that Smc6, together with Smc5, is involved in linking sister chromatids or homologous chromosomes, or bridging broken DNA ends. However, the precise molecular mechanism by which Smc6 promotes homologous recombination is not known yet.

The MRX complex in budding yeast, which is essential for Spo11-mediated meiotic recombination, is also required for DNA double strand break (DSB) repair in cells in the vegetative growth state (Alani et al., 1990; Ivanov et al., 1992; Johzuka et al., 1995; Bressan et al., 1999). Among the components of the MRX complex, Rad50 is structurally similar to the SMC family of proteins. Based on the architecture of the Rad50 dimer, it has been proposed that Rad50 links sister chromatids during homologous recombination, and DNA ends during non-homologous end-joining (Hopfner et al., 2002; Wiltzius et al., 2005). Thus, we were interested in knowing whether Rad50 is required for MMS induced homologous recombination in mitotic cells.

Since Rad50 is structurally similar to Smc6, we first examined the effect of deletion of RAD50 on the temperature-sensitivity of smc6-56 cells. In contrast to the results that deletion of SGS1, which encodes RecQ helicase, in smc6-9 (Torres-Rosell et al., 2005) or smc6-56 (data not shown) cells resulted in poor growth of these cells even in semi-permissive temperature, the viability of smc6-56 rad50 double mutants at the non-permissive temperature was similar to that of smc6-56 single mutants (Fig. 1A).

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Fig. 1. Lack of RAD50 does not dramatically affect the phenotype of smc6-56 mutant cells. (A) Survival curves for cells of the indicated genotypes at the restrictive temperature (37°C). Wild type cells (SCRMTL2) and smc6-56 cells (SCRMTs6g) were described previously (Onoda et al. 2004). rad50 (SCRrad50) and smc6-56 rad50 (SCRsmc6-56rad50) cells were constructed by PCR-based gene disruption of RAD50 in wild type and smc6-56 cells, respectively. Logarithmically growing cells were harvested, counted, and inoculated onto YPAD plates. The plates were incubated at 37°C for the indicated periods of time (days), and then cultured at 25°C for 3 days in total. Survival is expressed as a percentage of the number of colonies obtained after incubation at 25°C for 3 days, which was set as 100%. The data represents the average of two independent experiments; the bars indicate standard errors. (B) MMS sensitivity. Logarithmically growing cells were harvested, inoculated onto YPAD plates containing the indicated concentrations of MMS, and cultured at the semi-permissive temperature of 30°C for 3 days. The number of colonies on the plates was then counted. Survival is expressed as a percentage of the number of colonies obtained after incubation in the absence of MMS, which was set as 100%. The data represents the average of the two independent experiments; bars indicate standard errors.

We next examined DNA repair activity or damage-tolerance in cells exposed to methyl methanesulfonate (MMS). The single mutants smc6-56 and rad50 showed higher sensitivity to MMS compared with wild-type cells, while smc6-56 rad50 double mutants showed slightly higher sensitivity to MMS compared to rad50 mutants (Fig. 1B). Recently it was reported that Smc6 loading onto chromatin at DSB sites is dependent on the function of Mre11 (De Piccoli et al., 2006; Lindroos et al., 2006). Moreover, we recently found that Smc6 is required for MMS-induced sister chromatid recombination, as well as MMS-induced recombination between homologous chromosomes (Onoda et al., 2004). However, while MRX is essential for meiotic recombination between homologous chromosomes, mre11 mutants are proficient in both spontaneous recombination and UV-induced recombination between homologous chromosomes (Johzuka et al., 1995). UV-induced heteroallelic recombination between homologous chromosomes was increased in rad50 cells, similar to wild type cells (Fig. 2A). Interestingly, smc6-56 cells displayed no defect in UV-induced heteroallelic recombination (Fig. 2B), although these cells did show defects in MMS-induced heteroallelic recombination (Onoda et al., 2004).

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Fig. 2. UV-induced and MMS-induced heteroallelic recombination between homologous chromosomes. Strains with the MR101 background were constructed such that recombination between the heteroalleles his1-1 and his1-7 in diploid cells restored histidine prototrophy. Wild-type (MR101) and smc6-56 cells were described previously (Onoda et al. 2004). rad50 diploid cells in an MR101 background were generated for this study. (A), (B) UV-induced heteroallelic recombination. Wild-type (MR101), rad50 (A), or smc6-56 cells (B) were inoculated onto YPAD plates or SC plates lacking histidine (His), irradiated with the indicated doses of UV, and then incubated at 30°C for 3 days. (C) Wild-type (MR101) and rad50 cells were inoculated onto YPAD plates or SC plates lacking His and containing the indicated concentrations of MMS, then incubated at 30°C for 3 days. The number of His+ colonies per 106 survivors is presented, and the data is representative of at least three independent experiments. Bars indicate standard errors.

Based on results obtained with rad50, and xrs2 cells (Alani et al., 1990; Ivanov et al., 1992; Johzuka et al., 1995), it has been suggested that the MRX complex specifically executes homologous recombination between sister chromatids, but not between homologous chromosomes during vegetative growth (Symington, 2002). It has been proposed that Smc6 is recruited onto chromatin at DSB sites via the function of MRX complex (Lindroos et al., 2006). We were thus interested in whether Rad50 was required for MMS-induced recombination between homologous chromosomes, as is the case for Smc6. Indeed, we found that heteroallelic recombination between homologous chromosomes was not induced in rad50 cells upon exposure to MMS (Fig. 2C). Interestingly, mre11 mutant cells exhibit decreased frequencies of ionizing radiation-induced interhomologue recombination (Bressan et al., 1999). Taken together, it seems likely that MRX complex is involved in recombination between homologous chromosomes to repair DSBs in the cells in the vegetative growth state. Since most current models of the functions of Smc6 or the MRX complex relate their roles in sister-chromatid recombination (Symington, 2002; De Piccoli et al., 2006; Lindroos et al., 2006), our finding provide a new aspect for understanding the functions of Rad50 and Smc6 in recombination repair.

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Table 1. Yeast strains used in this study

We thank Dr. H. Ogawa for providing plasmid for disruption of RAD50 gene. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from The Ministry of Education, Science, Sports and Culture of Japan.