The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage

Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.     

The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase

Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2–7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S.cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G₁/S-boundary when quiescent human NB1–RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G₂ phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2–7 complexes.     

Mapping ubiquitination sites of S. cerevisiae Mcm10

Minichromosome maintenance protein (Mcm) 10 is a part of the eukaryotic replication machinery and highly conserved throughout evolution. As a multivalent DNA scaffold, Mcm10 coordinates the action of proteins that are indispensable for lagging strand synthesis, such as the replication clamp, proliferating cell nuclear antigen (PCNA). The binding between Mcm10 and PCNA serves an essential function during DNA elongation and is mediated by the ubiquitination of Mcm10. Here we map lysine 372 as the primary attachment site for ubiquitin on S. cerevisiae Mcm10. Moreover, we identify five additional lysines that can be ubiquitinated. Mutation of lysine 372 to arginine ablates ubiquitination of overexpressed protein and causes sensitivity to the replication inhibitor hydroxyurea in cells that are S-phase checkpoint compromised. Together, these findings reveal the high selectivity of the ubiquitination machinery that targets Mcm10 and that ubiquitination has a role in suppressing replication stress.     

Solution NMR Structure of the C-terminal DNA Binding Domain of Mcm10 Reveals a Conserved MCM Motif

The eukaryotic DNA replication protein Mcm10 associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Xenopus laevis (X) Mcm10 binds DNA via a highly conserved internal domain (ID) and a C-terminal domain (CTD) that is unique to higher eukaryotes. Although the structural basis of the interactions of the ID with DNA and polymerase α is known, little information is available for the CTD. We have identified the minimal DNA binding region of the XMcm10-CTD and determined its three-dimensional structure by solution NMR. The CTD contains a globular domain composed of two zinc binding motifs. NMR chemical shift perturbation and mutational analysis show that ssDNA binds only to the N-terminal (CCCH-type) zinc motif, whose structure is unique to Mcm10. The second (CCCC-type) zinc motif is not involved in DNA binding. However, it is structurally similar to the CCCC zinc ribbon in the N-terminal oligomerization domain of eukaryotic and archaeal MCM helicases. NMR analysis of a construct spanning both the ID and CTD reveals that the two DNA binding domains are structurally independent in solution, supporting a modular architecture for vertebrate Mcm10. Our results provide insight in the action of Mcm10 in the replisome and support a model in which it serves as a central scaffold through coupling of interactions with partner proteins and the DNA.     

Novel DNA Binding Properties of the Mcm10 Protein from Saccharomyces cerevisiae

The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a ∼1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21–24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20–50 nucleotides was ∼3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.MCM10 is a ubiquitous, conserved gene essential for DNA replication in eukaryotes. It was first discovered in yeast genetic screens designed to detect mutants defective in DNA synthesis and minichromosome maintenance (1, 2). In vivo, Mcm10 associates with chromatin and chromosomal replication origins in human cells (hMcm10), Xenopus laevis (XMcm10), Schizosaccharomyces pombe (SpMcm10), and Saccharomyces cerevisiae (ScMcm10) (3–6). In S. cerevisiae, initiation of chromosomal replication occurs at multiple specific sites known as autonomously replicating sequences (ARSs)² (7). Mutations in MCM10 enhance the loss rate of plasmids bearing specific ARSs (8), suggesting a function for ScMcm10 in initiation.In eukaryotic systems replication initiation is a cell cycle-regulated process characterized by a multistep sequential loading of ORC, Cdc6, Cdt1, and the Mcm2–7 complex onto the origin in G₁ to form the pre-RC complex. Binding of ORC, Cdc6p, and Cdt1p to chromatin is a prerequisite for the recruitment of Mcm2–7 (9, 10). The next step in the assembly of the initiation replication apparatus in S. cerevisiae involves protein kinases (Cdc28 and Cdc7/Dbf4), and recruitment of Mcm10, Cdc45, and the GINS complex. The mechanism for targeting Mcm10 to replications origins is unknown. However, recent studies in S. cerevisiae have shown that Mcm10 and Mcm2–7 physically interact (6, 11). It is now believed that in late G₁, chromatin-bound Mcm2–7 is responsible for the recruitment of Mcm10 presumably via protein-protein interactions (12). Prior studies in the Xenopus laevis system reached similar conclusions (4). Additional interactions of Mcm10 with other components of the pre-RC cannot be excluded (13).A key step in the initiation of replication is the local melting of an origin DNA sequence, which occurs at the G₁/S transition and throughout the S phase. The mechanism of this essential DNA-melting process is not understood. There is no evidence in S. cerevisiae that the assembled pre-RC complex leads to the melting of an origin DNA sequence. This unwinding may occur only following the recruitment of Mcm10, raising the possibility that Mcm10 is a key component of the initiation machinery responsible for this process. Results of a study in the Xenopus egg extract system (4), which showed that omission of XMcm10 blocks unwinding of plasmid DNA and initiation of DNA replication, are consistent with this notion. An additional function of Mcm10 in initiation is in the recruitment of Cdc45 to replication origins, presumably via Mcm10-Cdc45 physical interactions (5, 14). Cdc45 is believed to be important for the activation of replication origins and the assembly of the replication elongation complex (15). Upon initiation of DNA replication, ScMcm10 moves from the origin to origin-proximal sequences suggesting that ScMcm10 associates with moving replication forks (12) and is consistent with the observation that elevated temperatures cause pausing of replication forks in a mcm10-1 ts mutant (8). Both ScMcm10 and SpMcm10 interact with DNA polymerase α supporting the notion that replication fork movement requires Mcm10. ScMcm10 and polymerase α form a complex on and off the DNA in vivo (12). In S. pombe, SpMcm10 stimulates the activity of polymerase α in vitro and associates with a primase activity (16, 17) that has not been reported in other eukaryotes (18).Previous studies with Mcm10 in other systems showed that Mcm10 binds DNA. Using a filter binding assay Fien and Hurwitz (16) reported that SpMcm10 from S. pombe binds well to ssDNA but barely interacts (20-fold lower affinity) with dsDNA. It has been suggested that binding of SpMcm10 to ssDNA is important for the recruitment of polymerase α (16). Recently, it has been reported that a DNA binding activity is also associated with XMcm10 protein from X. laevis. Measurements of fluorescence anisotropy were used to show binding of XMcm10 to short, 25-nucleotide-long oligonucleotides (18). These studies have shown that XMcm10 has similar affinities for ssDNA and dsDNA. Unlike SpMcm10, which harbors a single DNA-binding domain in the N-terminal half of the protein, XMcm10 seems to contain two distinct domains for binding DNA. The biological implication of having two DNA-binding domains is not clear.It appears that there are differences in the quaternary structure of Mcm10 from different organisms. Although SpMcm10 and XMcm10 may be a homodimer in solution (17, 18), a recent electron microscopy study suggested that human hMcm10 has a hexameric ring structure (19). The same study reported that hMcm10 interacts with ssDNA but failed to bind dsDNA. The differences in structure and DNA binding properties may reflect differences in the function of Mcm10 in various organisms as well as in the protein preparations.Here we report, for the first time, the characterization of the DNA binding properties of purified Mcm10 from S. cerevisiae. We show that ScMcm10 forms a stable complex with dsDNA and ssDNA. In addition, we demonstrate that dsDNA longer than 50 bp sustains oligomerization of ScMcm10. The number of ScMcm10 molecules bound is directly proportional to the size of the dsDNA, suggesting that ScMcm10 is tightly packed on the dsDNA, perhaps in a head to tail oligomeric structure. In contrast to a 25-bp-long dsDNA, which supports the binding of a single copy of ScMcm10, ssDNA containing only 20 nucleotides may sustain binding of as many as three copies of ScMcm10, suggesting that a ScMcm10 complex composed of at least 3 subunits assembles on ssDNA. We believe that these distinct binding properties to dsDNA and ssDNA are important for the ScMcm10 functions in initiation, formation of replication forks, and the maintenance of replication fork progression during chromosomal DNA replication.     

Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex

Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly (“G₁-like”) and high affinity recruitment when CMG assembly takes place (“S-phase-like”). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus.     

Physical Interactions between Mcm10, DNA, and DNA Polymerase ��

Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase α (pol α), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol α. However, the mechanism by which Mcm10 interacts with pol α on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID·ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286–310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol α within the replication fork.To maintain their genomic integrity, cells must ensure complete and accurate DNA replication once per cell cycle. Consequently, DNA replication is a highly regulated and orchestrated series of molecular events. Multiprotein complexes assembled at origins of replication lead to assembly of additional proteins that unwind chromosomal DNA and synthesize nascent strands. The first event is the formation of a pre-replicative complex, which is composed of the origin recognition complex, Cdc6, Cdt1, and Mcm2–7 (for review, see Ref. 1). Initiation of replication at the onset of S-phase involves the activity of cyclin- and Dbf4-dependent kinases concurrent with recruitment of key factors to the origin. Among these, Mcm10 (2, 3) is recruited in early S-phase and is required for loading of Cdc45 (4). Mcm2–7, Cdc45, and the GINS complex form the replicative helicase (5–8). Origin unwinding is followed by loading of RPA,³ And-1/Ctf4, and pol α onto ssDNA (9–12). In addition, recruitment of Sld2, Sld3, and Dpb11/TopBP1 are essential for replication initiation (13, 14), and association of topoisomerase I, proliferating cellular nuclear antigen (PCNA), replication factor C, and the replicative DNA polymerases δ and ϵ completes the replisome (for review, see Ref. 15).Mcm10 is exclusive to eukaryotes and is essential to both initiation and elongation phases of chromosomal DNA replication (6, 8, 16). Mutations in Mcm10 in yeast result in stalled replication, cell cycle arrest, and cell death (2, 3, 17–19). These defects can be explained by the number of genetic and physical interactions between Mcm10 and many essential replication proteins, including origin recognition complex, Mcm2–7, and PCNA (3, 12, 20–24). In addition, Mcm10 has been shown to stimulate the phosphorylation of Mcm2–7 by Dbf4-dependent kinase in vitro (25). Thus, Mcm10 is an integral component of the replication machinery.Importantly, Mcm10 physically interacts with and stabilizes pol α and helps to maintain its association with chromatin (16, 26, 27). This is a critical interaction during replication because pol α is the only enzyme in eukaryotic cells that is capable of initiating DNA synthesis de novo. Indeed, Mcm10 stimulates the polymerase activity of pol α in vitro (28), and interestingly, the fission yeast Mcm10, but not Xenopus Mcm10, has been shown to exhibit primase activity (29, 30). Mcm10 is composed of three domains, the N-terminal (NTD), internal (ID), and C-terminal (CTD) domains (29). The NTD is presumably an oligomerization domain, whereas the ID and CTD both interact with DNA and pol α (29). The CTD is not found in yeast, whereas the ID is highly conserved among all eukaryotes. The crystal structure of Mcm10-ID showed that this domain is composed of an oligonucleotide/oligosaccharide binding (OB)-fold and a zinc finger motif, which form a unified DNA binding platform (31). An Hsp10-like motif important for the interaction with pol α has been identified in the sequence of Saccharomyces cerevisiae Mcm10-ID (16, 26).DNA pol α-primase is composed of four subunits: p180, p68, p58, and p48. The p180 subunit possesses the catalytic DNA polymerase activity, and disruption of this gene is lethal (32, 33). p58 and p48 form the DNA-dependent RNA polymerase (primase) activity (34, 35), whereas the p68 subunit has no known catalytic activity but serves a regulatory role (36, 37). Pol α plays an essential role in lagging strand synthesis by first creating short (7–12 nucleotide) RNA primers followed by DNA extension. At the critical length of ∼30 nucleotides, replication factor C binds to the nascent strand to displace pol α and loads PCNA with pols δ and ϵ (for review, see Ref. 38).The interaction between Mcm10 and pol α has led to the suggestion that Mcm10 may help recruit the polymerase to the emerging replisome. However, the molecular details of this interaction and the mechanism by which Mcm10 may recruit and stabilize the pol α complex on DNA has not been investigated. Presented here is the high resolution structure of the conserved Mcm10-ID bound to ssDNA together with NMR chemical shift perturbation competition data for pol α binding in the presence of ssDNA. Collectively, these data demonstrate a shared binding site for DNA and pol α in the OB-fold cleft of Mcm10-ID, with a preference for ssDNA over pol α. In addition, we have mapped the Mcm10-ID binding site on pol α to a 24-residue segment of the N-terminal domain of p180. Based on these results, we propose Mcm10 helps to recruit pol α to origins of replication by a molecular hand-off mechanism.     

Ancient diversification of eukaryotic MCM DNA replication proteins

Background  

Yeast and animal cells require six mini-chromosome maintenance proteins (Mcm2-7) for pre-replication complex formation, DNA replication initiation and DNA synthesis. These six individual MCM proteins form distinct heterogeneous subunits within a hexamer which is believed to form the replicative helicase and which associates with the essential but non-homologous Mcm10 protein during DNA replication. In contrast Archaea generally only possess one MCM homologue which forms a homohexameric MCM helicase. In some eukaryotes Mcm8 and Mcm9 paralogues also appear to be involved in DNA replication although their exact roles are unclear.     

Xenopus Mcm10 is a CDK-substrate required for replication fork stability

During S phase, following activation of the S phase CDKs and the DBF4-dependent kinases (DDK), double hexamers of Mcm2-7 at licensed replication origins are activated to form the core replicative helicase. Mcm10 is one of several proteins that have been implicated from work in yeasts to play a role in forming a mature replisome during the initiation process. Mcm10 has also been proposed to play a role in promoting replisome stability after initiation has taken place. The role of Mcm10 is particularly unclear in metazoans, where conflicting data has been presented. Here, we investigate the role and regulation of Mcm10 in Xenopus egg extracts. We show that Xenopus Mcm10 is recruited to chromatin late in the process of replication initiation and this requires prior action of DDKs and CDKs. We also provide evidence that Mcm10 is a CDK substrate but does not need to be phosphorylated in order to associate with chromatin. We show that in extracts depleted of more than 99% of Mcm10, the bulk of DNA replication still occurs, suggesting that Mcm10 is not required for the process of replication initiation. However, in extracts depleted of Mcm10, the replication fork elongation rate is reduced. Furthermore, the absence of Mcm10 or its phosphorylation by CDK results in instability of replisome proteins on DNA, which is particularly important under conditions of replication stress.     

Ctf4p facilitates Mcm10p to promote DNA replication in budding yeast

Ctf4p (chromosome transmission fidelity) has been reported to function in DNA metabolism and sister chromatid cohesion in Saccharomyces cerevisiae. In this study, a ctf4^S143F mutant was isolated from a yeast genetic screen to identify replication-initiation proteins. The ctf4^S143F mutant exhibits plasmid maintenance defects which can be suppressed by the addition of multiple origins to the plasmid, like other known replication-initiation mutants. We show that both ctf4^S143F and ctf4Δ strains have defects in S phase entry and S phase progression at the restrictive temperature of 38 °C. Ctf4p localizes in the nucleus throughout the cell cycle but only starts to bind chromatin at the G1/S transition and then disassociates from chromatin after DNA replication. Furthermore, Ctf4p interacts with Mcm10p physically and genetically, and the chromatin association of Ctf4p depends on Mcm10p. Finally, deletion of CTF4 destabilizes Mcm10p and Pol α in both mcm10-1 and MCM10 cells. These data indicate that Ctf4p facilitates Mcm10p to promote the DNA replication.     

Ubc4 and Not4 Regulate Steady-State Levels of DNA Polymerase-�� to Promote Efficient and Accurate DNA Replication

The accurate duplication of chromosomal DNA is required to maintain genomic integrity. However, from an evolutionary point of view, a low mutation rate during DNA replication is desirable. One way to strike the right balance between accuracy and limited mutagenesis is to use a DNA polymerase that lacks proofreading activity but contributes to DNA replication in a very restricted manner. DNA polymerase-α fits this purpose exactly, but little is known about its regulation at the replication fork. Minichromosome maintenance protein (Mcm) 10 regulates the stability of the catalytic subunit of pol-α in budding yeast and human cells. Cdc17, the catalytic subunit of pol-α in yeast, is rapidly degraded after depletion of Mcm10. Here we show that Ubc4 and Not4 are required for Cdc17 destabilization. Disruption of Cdc17 turnover resulted in sensitivity to hydroxyurea, suggesting that this pathway is important for DNA replication. Furthermore, overexpression of Cdc17 in ubc4 and not4 mutants caused slow growth and synthetic dosage lethality, respectively. Our data suggest that Cdc17 levels are very tightly regulated through the opposing forces of Ubc4 and Not4 (destabilization) and Mcm10 (stabilization). We conclude that regular turnover of Cdc17 via Ubc4 and Not4 is required for proper cell proliferation.     

Ciprofloxacin is an inhibitor of the Mcm2-7 replicative helicase

Most currently available small molecule inhibitors of DNA replication lack enzymatic specificity, resulting in deleterious side effects during use in cancer chemotherapy and limited experimental usefulness as mechanistic tools to study DNA replication. Towards development of targeted replication inhibitors, we have focused on Mcm2-7 (minichromosome maintenance protein 2–7), a highly conserved helicase and key regulatory component of eukaryotic DNA replication. Unexpectedly we found that the fluoroquinolone antibiotic ciprofloxacin preferentially inhibits Mcm2-7. Ciprofloxacin blocks the DNA helicase activity of Mcm2-7 at concentrations that have little effect on other tested helicases and prevents the proliferation of both yeast and human cells at concentrations similar to those that inhibit DNA unwinding. Moreover, a previously characterized mcm mutant (mcm4chaos3) exhibits increased ciprofloxacin resistance. To identify more potent Mcm2-7 inhibitors, we screened molecules that are structurally related to ciprofloxacin and identified several that compromise the Mcm2-7 helicase activity at lower concentrations. Our results indicate that ciprofloxacin targets Mcm2-7 in vitro, and support the feasibility of developing specific quinolone-based inhibitors of Mcm2-7 for therapeutic and experimental applications.     

Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus

The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing. Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea. The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue. The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro. The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA. Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA. The purified Orc/Cdc6 homologue was also able to bind DNA. Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates. Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap. The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer.     

Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion

At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis. The cdc24⁺ gene encodes a 501-amino-acid protein with no significant homology to any known proteins. The temperature-sensitive cdc24 mutant is effectively rescued by pcn1⁺, rfc1⁺ (a fission yeast homologue of RFC1), and hhp1⁺, which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively. The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule. In addition, cdc24⁺ genetically interacts with the gene encoding the catalytic subunit of DNA polymerase , which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10. These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.     

Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components

The CMG complex composed of Mcm2-7, Cdc45 and GINS is postulated to be the eukaryotic replicative DNA helicase, whose activation requires sequential recruitment of replication proteins onto Mcm2-7. Current models suggest that Mcm10 is involved in assembly of the CMG complex, and in tethering of DNA polymerase α at replication forks. Here, we report that Mcm10 is required for origin DNA unwinding after association of the CMG components with replication origins in fission yeast. A combination of promoter shut-off and the auxin-inducible protein degradation (off-aid) system efficiently depleted cellular Mcm10 to <0.5% of the wild-type level. Depletion of Mcm10 did not affect origin loading of Mcm2-7, Cdc45 or GINS, but impaired recruitment of RPA and DNA polymerases. Mutations in a conserved zinc finger of Mcm10 abolished RPA loading after recruitment of Mcm10. These results show that Mcm10, together with the CMG components, plays a novel essential role in origin DNA unwinding through its zinc-finger function.