In eukaryotes, three pairs of structural-maintenance-of-chromosome (SMC) proteins are found in conserved multisubunit protein complexes required for chromosomal organization. Cohesin, the Smc1/3 complex, mediates sister chromatid cohesion while two condensin complexes containing Smc2/4 facilitate chromosome condensation. Smc5/6 scaffolds an essential complex required for homologous recombination repair. We have examined the response of smc6 mutants to the inhibition of DNA replication. We define homologous recombination-dependent and -independent functions for Smc6 during replication inhibition and provide evidence for a Rad60-independent function within S phase, in addition to a Rad60-dependent function following S phase. Both genetic and physical data show that when forks collapse (i.e., are not stabilized by the Cds1 Chk2 checkpoint), Smc6 is required for the effective repair of resulting lesions but not for the recruitment of recombination proteins. We further demonstrate that when the Rad60-dependent, post-S-phase Smc6 function is compromised, the resulting recombination-dependent DNA intermediates that accumulate following release from replication arrest are not recognized by the G 2 /M checkpoint.The eukaryotic structural-maintenance-of-chromosome (SMC) proteins form heterodimers that are the cores of several evolutionarily conserved multisubunit protein complexes. An Smc1/3 complex mediates sister chromatid cohesion during both mitosis and meiosis and is known as cohesin. Chromosome condensation requires two related Smc2/4-based complexes known as condensins (17). Smc5 and Smc6 form a complex along with six non-Smc components (Nse1 to Nse6). An additional protein, Rad60, has been shown to associate with the Smc5/6-Nse1-6 core complex nonstoichiometrically. In Schizosaccharomyces pombe, Smc5, Smc6, and Nse1-4 complex components are essential and loss of function results in chromosomal fragmentation that requires passage through S phase. Hypomorphic smc6, nse1, nse2, nse3, and nse4 mutants are all defective in homologous recombination repair (HRR) (5,14,16,27,33,37,39,45,54). Similar observations have been made for Saccharomyces cerevisiae, where compromising the Smc5/6 complex function interferes with ribosomal DNA (rDNA) segregation and sister chromatid exchange (11,19,53). In contrast, S. pombe Nse5 and Nse6 are nonessential. nse5 and nse6 null mutants grow slowly and exhibit repair defects similar to those of the smc5/6 complex hypomorphic mutants (46).Of the non-SMC components, Nse1 contains a RING domain typical of ubiquitin E3 ligases, although its substrates and activity remain unidentified. Nse2 is a SUMO E3 ligase and directs the sumoylation of multiple substrates, including S. pombe Smc6 and S. cerevisiae and human Smc5. Nse3 is a member of the MAGE (melanoma antigen-encoding gene) superfamily and, like Nse4, is a conserved protein of an unknown function (2,33,45,47,50,56). The core subunit components localize to the nucleus together with the noncore component Rad60, which has a C-terminal ubiqu...

The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome mis-segregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination-dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome-wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage-induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination-competent conformation primed for replication restart.

Members of the RecQ helicase subfamily are mutated in several human genomic instability syndromes, such as Bloom, Werner, and Rothmund-Thomson syndromes. We show that Rqh1, the single Schizosaccharomyces pombe homologue, is a 3-to-5 helicase and exists with Top3 in a high-molecular-weight complex. top3 deletion is inviable, and this is suppressed by concomitant loss of rqh1 helicase activity or loss of recombination functions. This is consistent with RecQ helicases in other systems. By using epistasis analysis of the UV radiation sensitivity and by analyzing the kinetics of Rhp51 (Rad51 homologue), Rqh1, and Top3 focus formation in response to UV in synchronized cells, we identify the first evidence of a function for Rqh1 and Top3 in the repair of UV-induced DNA damage in G 2 . Our data provide evidence that Rqh1 functions after Rad51 focus formation during DNA repair. We also identify a function for Rqh1 upstream of recombination in an Rhp18-dependent (Rad18 homologue) pathway. The model that these data allow us to propose helps to reconcile different interpretations of RecQ family helicase function that have arisen between work based on the S. pombe system and models based on studies of Saccharomyces cerevisiae SGS1 suggesting that RecQ helicases act before Rad51.