Mcm10 deficiency causes defective-replisome-induced mutagenesis and a dependency on error-free postreplicative repair

Abstract

Mcm10 is a multifunctional replication factor with reported roles in origin activation, polymerase loading, and replication fork progression. the literature supporting these variable roles is controversial, and it has been debated whether Mcm10 has an active role in elongation. Here, we provide evidence that the mcm10-1 allele confers alterations in DNA synthesis that lead to defective-replisome-induced mutagenesis (DRIM). Specifically, we observed that mcm10-1 cells exhibited elevated levels of pCNA ubiquitination and activation of the translesion polymerase, pol-ζ. Whereas translesion synthesis had no measurable impact on viability, mcm10-1 mutants also engaged in error-free postreplicative repair (pRR), and this pathway promoted survival at semi-permissive conditions. Replication gaps in mcm10-1 were likely caused by elongation defects, as dbf4-1 mutants, which are compromised for origin activation did not display any hallmarks of replication stress. Furthermore, we demonstrate that deficiencies in priming, induced by a pol1-1 mutation, also resulted in DRIM, but not in error-free pRR. Similar to mcm10-1 mutants, DRIM did not rescue the replication defect in pol1-1 cells. thus, it appears that DRIM is not proficient to fill replication gaps in pol1-1 and mcm10-1 mutants. Moreover, the ability to correctly prime nascent DNA may be a crucial prerequisite to initiate error-free pRR.

Slx5/Slx8 Promotes Replication Stress Tolerance by Facilitating Mitotic Progression

“…At higher temperatures, however, when cells accumulated more severe DNA damage (Becker et al, 2014), the majority of mcm10-1 and mcm10-1 slx5Δ mutants remained arrested in G2/M (Figure 6B), consistent with a more robust Rad53 activation observed at 35°C compared to 33°C (Figures 6C and D). Hyper-phosphorylation of Rad53 observed at 35°C in mcm10-1 mutants was slightly diminished when SLX5 was knocked out, implying that Slx5 contributes to checkpoint signaling under extreme DNA damage conditions (Figure 6D).…”

“…Heat-induced depletion of Mcm10 causes replication stress displaying the typical hallmarks of Rad53 phosphorylation and PCNA ubiquitination (Becker et al, 2014). SGA analysis identified SLX5 and SLX8 as top hits that exhibited synthetic sickness with the mcm10-1 allele at 30°C (Thu and Bielinsky, 2013, 2014).…”

“…The functional RING domain of Slx5 was necessary to reverse the growth defect of mcm10-1 slx5Δ cells (Figure 1A), arguing that the catalytic function of Slx5/8 was required to confer resistance to replication stress (Xie et al, 2007). However, with increasing temperatures, SLX5 expression gradually lost the ability to rescue the viability of mcm10-1 slx5Δ mutants (Figure 1A), likely because the cells accumulated too much DNA damage (Becker et al, 2014). Re-expressing MCM10 under the control of its endogenous promoter fully rescued the growth defect of the double mutants at all temperatures (Figure 1B).…”

Mcm10 is required for oogenesis and early embryogenesis in Drosophila

“…This is contrary to the data generated in most single celled systems which suggest that Mcm10 is required for multiple stages of DNA replication (Thu and Bielinsky, 2013). Data from S. cerevisiae has demonstrated that DNA replication can occur in Mcm10 deficient backgrounds through a dependence on error-free post-replicative repair, although at the cost of higher mutation rates (Becker et al, 2014). The observed adult survivorship in these mutants does not necessarily equate to normal S-phase progression, the presence of endogenous chromatin states, or a natural rate of mutation accumulation.…”

“…Current evidence supports the idea that Mcm10 travels with the processing replisome (Aparicio et al, 1997; Das-Bradoo et al, 2006), and modulates chromatin dynamics (Apger et al, 2010; Liachko and Tye, 2009). Mcm10 has also been demonstrated to promote genomic stability through extensive interplay with DNA damage repair mechanisms (Alver et al, 2014; Becker et al, 2014; Thu and Bielinsky, 2014). Despite the wealth of data describing the functions of Mcm10 in various steps in DNA metabolism, conflicting results between model systems have left the essential functions of Mcm10 highly debated (Thu and Bielinsky, 2013).…”