The Smc5-Smc6 complex

The third of the Smc complexes, Smc5-Smc6, was first identified in Schizosaccharomyces pombe (Lehmann et al., 1995). The as yet unnamed Smc5-Smc6 complex, together with six non-Smc elements (Nse1 to Nse6), is mainly required for DNA damage repair and G2 checkpoint maintenance (Verkade et al., 1999) after treatment with a broad range of DNA damaging agents, including UV, IR, MMS and cis-platin (Fousteri and Lehmann, 2000; Lehmann et al., 1995; Taylor et al., 2001). All genes of the Smc5-Smc6 complex are essential for viability in S. cerevisiae. On the contrary, the S. pombe Nse5 and Nse6 are not required for viability. Surprisingly, the human (Potts et al., 2006; Potts and Yu, 2005), Arabidopsis thaliana (Watanabe et al., 2009) and chicken (Stephan et al., 2011a) Smc5-Smc6 complexes are not essential for cell proliferation.

Similarly to the cohesin and condensin complexes, the Smc5 and Smc6 proteins heterodimerise through hinge interactions to form a scaffold for a high molecular weight complex. The non-Smc elements bind to this scaffold to form a functional Smc6 complex. Among the non-Smc elements of the Smc5-Smc6 complex, the Nse1 protein contains a zing finger domain similar to E3 ubiquitin ligases but no activity has yet been observed in vivo (McDonald et al., 2003). Recent work by Pebernard et al. revealed no ubiquitin ligase activity for Nse1 in vitro. The authors suggested that the zinc finger domain is required for formation of the Nse1-Nse3-Nse4 subcomplex (Pebernard et al., 2008b).

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However, Nse1 showed a robust activity in the presence of Nse3, indicating that Nse1 is a functional E3 ubiquitin ligase (Doyle et al., 2010). Nse2/MMS21 is an active Siz/PIAS E3 SUMO ligase that SUMOylates many cellular targets (see section 1.10.4). Nse3 is a member of the MAGE (melanoma associated antigen) family of unknown function (Pebernard et al., 2004). Nse4 has been identified as the kleisin element of the Smc5-Smc6 complex, whose function is to bridge the Smc5-Smc6 heterodimer heads (Palecek et al., 2006). It contains the kleisin-like domain composed of a helix-turn-helix motif, which is also found in other members of the family, including Scc1 and CAP-H (Palecek et al., 2006;

Pebernard et al., 2004). Nse5 does not contain any known domains and Nse6 has been identified as HEAT (Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the yeast PI3-kinase TOR1) repeat protein (Palecek et al., 2006; Pebernard et al., 2006). The Nse5 and Nse6 proteins form another subcomplex and like the Nse1-Nse3-Nse4 heterotrimer, bind to the heads or arms of the Smc5-Smc6 complex in S. pombe (Palecek et al., 2006; Pebernard et al., 2006) and to hinges in S. cerevisiae (Duan et al., 2009b).

Chromatin immunoprecipitation (ChiP) experiments in budding and fission yeast revealed different chromosomal localisations of the Smc5-Smc6 complex, with enrichment at telomeres, centromeres and rDNA gene clusters (Ampatzidou et al., 2006; Lindroos et al., 2006; Pebernard et al., 2008c; Torres-Rosell et al., 2005a). Defective HR at the repetitive DNA sequences can result in gross chromosomal rearrangements such as deletions and insertions at these loci.

It has been proposed that the localisation of the Smc5-Smc6 complex on these sequences regulates their status through recombination (Torres-Rosell et al., 2005b; Zhao and Blobel, 2005). In addition, yeast Smc5-Smc6 mutants show increased telomere shortening and in human cells, the Smc5-Smc6 is required for recombination-dependent alternative telomere lengthening (ALT) process (Chavez et al., 2010b; Potts and Yu, 2007). DNA repair and segregation at ribosomal loci is also mediated by the Smc5-Smc6 complex (Torres-Rosell et al., 2005a; Torres-Rosell et al., 2005b; Torres-Rosell et al., 2007).

The functions of the Smc5-Smc6 complex are considered to be in DNA repair. The S. pombe Rad18/Smc6 gene is required for maintenance of G2

checkpoint and repair after DNA damage (Lehmann et al., 1995; Verkade et al.,

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1999). The Smc5-Smc6 complex plays a role in the restart of the stalled replication forks by regulating recombination (Ampatzidou et al., 2006; Irmisch et al., 2009). Studies in yeast, chicken and human cells have revealed that the Smc5-Smc6 complex is required for efficient homologous recombinational repair (Lehmann et al., 1995; Potts et al., 2006; Stephan et al., 2011a; Verkade et al., 1999). In epistasis analyses experiments, combined mutation of Smc5 and HR genes, such as Rad51 or Rad54 does not enhance sensitivity towards IR, UV or MMS, further confirming a HR role for the complex (Ampatzidou et al., 2006;

Bermúdez-López et al., 2010; Lehmann et al., 1995; McDonald et al., 2003;

Stephan et al., 2011a). Consistent with such a role for Smc5-Smc6, depletion of the NHEJ gene Ku70 in chicken Smc5-deficient cells caused further sensitisation of Smc5 mutants to IR-induced DNA damage (Stephan et al., 2011a).

Recruitment of the Rad51 and Rad54 proteins to DSB is unaffected in chicken and fission yeast mutants of the Smc5-Smc6 complex, suggesting its activity in late stages of HR (Ampatzidou et al., 2006; Stephan et al., 2011a). Extensive studies in budding and fission yeast using 2D gel electrophoresis revealed a significant increase in the X-shaped molecules in the absence of functional Smc5-Smc6 complex before and after MMS or HU treatment (Ampatzidou et al., 2006; Branzei et al., 2006; Chavez et al., 2010a; Chavez et al., 2010b; Chen et al., 2009; Choi et al., 2010). The nature of these X-shaped molecules has not been confirmed yet but a large body of data suggests that this are recombination intermediates (Ampatzidou et al., 2006; Bermúdez-López et al., 2010; Branzei et al., 2006; Choi et al., 2010). This is also associated with aberrant mitosis as defined by chromosome mis-segregation, premature septation in the presence of unrepaired or not completely replicated DNA (‘cut’ phenotype), nuclear segregation and unequal DNA mass separation between mother and daughter cells (Ampatzidou et al., 2006; Chavez et al., 2010a; Chavez et al., 2010b; Chen et al., 2009; Choi et al., 2010; Lehmann et al., 1995; Verkade et al., 1999).

Together, these observations indicate that the Smc5-Smc6 complex acts in the late stages of HR. The loss of Smc5-Smc6 complex leads to impaired HR and the accumulation of lethal DNA repair intermediates. The X-shaped DNA molecules arising in smc6-1 and mms21-sp can be removed and the mitotic aberrations reversed by restoration of the Smc5-Smc6 complex through expression of either

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wild-type Smc6 or Nse2 in these mutants (Bermúdez-López et al., 2010; Branzei et al., 2006; Chavez et al., 2010b). Additional evidence for a HR function of the Smc5-Smc6 complex comes from experiments where deletion of HR factors such as MphI/FANCM helicase in smc6-9 and mms21-sp, Shu complex in smc6-P4 and smc6-56, the post replicative repair protein MMS2 in smc6-P4 and smc6-56 or overexpression of the 6-BRCT containing protein Brc1 in smc6-74, reverse hypersensitivity towards DNA damaging agents such as MMS, HU and UV (Ampatzidou et al., 2006; Chavez et al., 2010a; Choi et al., 2010; Lee et al., 2007; Sheedy et al., 2005). These findings clearly indicate that the Smc5-Smc6 complex is required for resolution of specific HR intermediates which can be removed by other HR factors acting upstream of the Smc5-Smc6 complex or by shifting the repair balance towards alternative HR repair pathways.

In HeLa cells but not in budding and fission yeast, cohesin recruitment to DSB is mediated by the Smc5-Smc6 complex (De Piccoli et al., 2006; Outwin et al., 2009; Potts et al., 2006). As the Smc1-Smc3 complex is required for local sister chromatid cohesion around the double strand break, these data suggest that Smc5-Smc6 complex may regulate cohesion to facilitate DSB repair (Ström et al., 2004). Another group reported that upon Nse2 and Smc5 siRNA mediated knockdown, HeLa cells showed severe loss of sister chromatid cohesion (Behlke-Steinert et al., 2009). Our group found that depletion of chicken Smc5 in DT40 cells results in increased inter-sister chromatid distances before and after DSB induction. We also observed no further cohesion loss in double Scc1 -/-/-Smc5^- mutants, what demonstrates that Smc5-Smc6 complex is epistatic to cohesin in cohesion maintenance (Stephan et al., 2011a). Another group found that in fission yeast, in the absence of functional Smc5-Smc6 complex, the cohesin complex is retained at chromosome arms resulting in chromosome segregation defects (Outwin et al., 2009). Recruitment of cohesin to DSB by Smc5-Smc6 is recognised as a very early event in the HR process. Similarly, the localisation of the ‘early’ HR factor Rad52, which is required for recombination at stalled replication forks is Smc5-Smc6 dependent (Irmisch et al., 2009). We found that Smc5-deficient cells efficiently mobilise Rad51 to IR-induced DNA repair foci (Stephan et al., 2011a). These findings indicate dual functions of the Smc5-Smc6 complex at different stages of the HR pathway. It is hard to explain

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how a single complex could be involved at various steps of DNA repair where different activities are required. Therefore many groups have proposed that the Smc5-Smc6 complex is involved in the regulation of global chromatin conformation, like other Smc complexes rather than acting at specific steps of DNA repair (Ampatzidou et al., 2006; Torres-Rosell et al., 2007).