Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

Abstract

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...

Human Stn1 protects telomere integrity by promoting efficient lagging-strand synthesis at telomeres and mediating C-strand fill-in

“…During replication, polα is brought to replication origins via interaction with Mcm10 and And-1/Ctf4 to initiate lagging-strand synthesis [45][46][47]. In the late S/G2 phase, polα is recruited back to telomeres [48] (Figure 6), presumably for filling in C-strands.…”

Physical Interactions between Mcm10, DNA, and DNA Polymerase α

“…This follows a common strategy for numerous modular proteins involved in DNA processing; there is a significant kinetic advantage to deconstructing protein interactions into two or more weak binding sites (68). The recruitment of pol ␣ to origins of replication by Mcm10 would be a significant step to signal nascent DNA synthesis and contribute to fork stability (6,12,16,24,26,27,29,71). Indeed, Mcm10 has been shown to be necessary for pol ␣ loading onto chromatin (4).…”

“…Importantly, Mcm10 physically interacts with and stabilizes pol ␣ and helps to maintain its association with chromatin (16,26,27). This is a critical interaction during replication because pol ␣ is the only enzyme in eukaryotic cells that is capable of initiating DNA synthesis de novo.…”

Assembly of the Cdc45-Mcm2–7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins

“…Although neutralizing antibodies against TopBP1, the human homologue of Dpb11/Cut5, were reported to efficiently inhibit replicative DNA synthesis in HeLa cell nuclei in vitro (21), the major role of human TopBP1 seems to be in the S phase checkpoint control (22). RECQL4, which is mutated in the Rothmund-Thomson Syndrome, and Mcm10 were shown to be required for the initiation of DNA replication in human cells, but their roles in the initiation process are still unknown (23)(24)(25)(26). Ctf4/And-1, which is required for sister chromatid cohesion in yeasts (27), was also shown to be essential for the chromatin binding of DNA polymerase ␣ and for the initiation of DNA replication in mammalian cells (25).…”