Role of the SMC5/6 complex in DNA replication and DNA damage repair
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AbstractThe structural maintenance of chromosome (SMC) proteins form the core of Cohesin, Condensin and the Smc5/6 complex, which are essential for organization and metabolism of chromosomes during the cell cycle. The Smc5/6 complex is implicated a role in non-essential homologous recombination-mediated (HR) DNA repair. Inactivating Smc5/6 (via temperature-sensitivity mutant) in the S phase, but not in the G2 phase, causes mitotic failure. Hence, we hypothesize that the Smc5/6 complex has an major role in DNA replication. We analyzed the genome-wide DNA replication temporal program in different genetic backgrounds. Mapping replication fork-associated ssDNA in WT and rad53 cells in the presence of replication stress allowed us to separate early and late replication origins. Using this method, we revealed a strain background difference of origin usage in A364a and W303. We then studied the genome-wide replication dynamics in a hypomorphic mutant, smc6-P4, using a density transfer coupled with microarray method. Overall replication dynamics of the mutant are similar to that of the WT cells with exceptions in the early S phase. However, we captured a difference in the replication profile of an early S phase sample in the mutant, prompting the hypothesis that the mutant incorporates ribonucleotides and/or accumulates single-stranded DNA gaps during replication. We then tested whether we can exacerbate the DNA replication stress hypersensitivity of the smc6 mutant by inhibiting ribonucleotides excision repair (RER) pathway. Contrary to our expectation, impairment of ribonucleotide excision repair, as well as virtually all other DNA repair pathways, alleviated smc6 mutant’s hypersensitivity to induced replication stress. We propose that nucleotide incision with impaired Smc5/6 complex has a more disastrous outcome than the damage itself. We found that the smc6 mutant has a significant increase in ssDNA level under replication stress but inhibiting RER pathway decreases it. This result suggests that Smc5/6 complex prevents unsupervised ssDNA formation during DNA replication as a result of damage incision repair. We reason that excess ssDNA in the Smc5/6 defective cells may subsequently engage in toxic recombination. Our study thus provides novel perspectives for the role of the Smc5/ 6 complex during DNA replication.
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