Mcm10 is a conserved eukaryotic DNA replication factor whose function has remained elusive. We report here that Mcm10 binding to replication origins in budding yeast is cell cycle regulated and dependent on the putative helicase, Mcm2-7. Mcm10 is also an essential component of the replication fork. A fraction of Mcm10 binds to DNA, as shown by histone association assays that allow for the study of chromatin binding in vivo. However, Mcm10 is also required to maintain steady-state levels of DNA polymerase-alpha (polalpha). In temperature-sensitive mcm10-td mutants, depletion of Mcm10 during S phase results in degradation of the catalytic subunit of polalpha, without affecting other fork components such as Cdc45. We propose that Mcm10 stabilizes polalpha and recruits the complex to replication origins. During elongation, Mcm10 is required for the presence of polalpha at replication forks and may coordinate DNA synthesis with DNA unwinding by the Mcm2-7 complex.

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.

Summary Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase α, and PCNA. The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a novel DNA binding architecture composed of an OB-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, pol α, and DNA interactions onto the crystal structure provides insight into how Mcm10 may coordinate protein and DNA binding within the replisome.

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Sites of DNA synthesis initiation have been detected at the nucleotide level in a yeast origin of bidirectional replication with the use of replication initiation point mapping. The ARS1 origin of Saccharomyces cerevisiae showed a transition from discontinuous to continuous DNA synthesis in an 18-base pair region (nucleotides 828 to 845) from within element B1 toward B2, adjacent to the binding site for the origin recognition complex, the putative initiator protein.

Initiation sites for DNA synthesis in the chromosomal autonomously replicating sequence (ARS)1 of Saccharomyces cerevisiae were detected at the nucleotide level. The transition from discontinuous to continuous synthesis defines the origin of bidirectional replication (OBR), which mapped adjacent to the origin recognition complex binding site. To ascertain which sites represented starts for leading or lagging strands, we characterized DNA replication from ARS1 in a cdc9 (DNA ligase I) mutant, defective for joining Okazaki fragments. Leading strand synthesis in ARS1 initiated at only a single site, the OBR. Thus, replication in S. cerevisiae is not initiated stochastically by choosing one out of multiple possible sites but, rather, is a highly regulated process with one precise start point.

In Saccharomyces cerevisiae, minichromosome maintenance protein (Mcm) 10 interacts with DNA polymerase (pol)-␣ and functions as a nuclear chaperone for the catalytic subunit, which is rapidly degraded in the absence of Mcm10. We report here that the interaction between Mcm10 and pol-␣ is conserved in human cells. We used a small interfering RNA-based approach to deplete Mcm10 in HeLa cells, and we observed that the catalytic subunit of pol-␣, p180, was degraded with similar kinetics as Mcm10, whereas the regulatory pol-␣ subunit, p68, remained unaffected. Simultaneous loss of Mcm10 and p180 inhibited S phase entry and led to an accumulation of already replicating cells in late S/G2 as a result of DNA damage, which triggered apoptosis in a subpopulation of cells. These phenotypes differed considerably from analogous studies in Drosophila embryo cells that did not exhibit a similar arrest. To further dissect the roles of Mcm10 and p180 in human cells, we depleted p180 alone and observed a significant delay in S phase entry and fork progression but little effect on cell viability. These results argue that cells can tolerate low levels of p180 as long as Mcm10 is present to "recycle" it. Thus, human Mcm10 regulates both replication initiation and elongation and maintains genome integrity. INTRODUCTIONDNA replication is a highly regulated process in which multiprotein complexes generate an exact copy of a cell's DNA. These multiprotein complexes are assembled into replication forks. Fork assembly takes place at replication origins that are spread throughout the genome in all eukaryotic cells. The first step is the formation of a prereplicative complex (pre-RC) (Diffley et al., 1994), which is subsequently transformed into a preinitiation complex, and finally into a pair of functional replication forks that have the ability to unwind parental DNA and synthesize new daughter strands (Mendez and Stillman, 2003). DNA unwinding is likely catalyzed by the minichromosome maintenance (Mcm) 2-7 complex (Aparicio et al., 1997;Labib et al., 2000;Shechter et al., 2004) in conjunction with two coactivators, Cdc45 and GINS (Pacek and Walter, 2004;Gambus et al., 2006;Moyer et al., 2006;Pacek et al., 2006). The association of Cdc45 and GINS with DNA is interdependent (Kubota et al., 2003;Takayama et al., 2003), and it requires yet another factor, Mcm10 (Wohlschlegel et al., 2002;Gregan et al., 2003;Sawyer et al., 2004). Despite its name, Mcm10 is not a member of the MCM protein family, although it was isolated in the same genetic screen in budding yeast that identified the MCM2-7 genes (Solomon et al., 1992;Merchant et al., 1997).Mcm10 is a constitutively nuclear DNA binding protein (Merchant et al., 1997;Burich and Lei, 2003) that is essential in yeast (Burich and Lei, 2003). Besides its role in DNA replication, Mcm10 has also been implicated in transcriptional silencing (Liachko and Tye, 2005). It is important to note that the general protein domain structure of Mcm10 is not unique in Saccharomyces cerevisiae, but it is highly con...

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