Structural insights into the Cdt1-mediated MCM2-7 chromatin loading.

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410-440)/MCM6(708-821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2-7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2-7, our results suggest that the MCM2-7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2-7 chromatin loading.

Ploidy dictates repair pathway choice under DNA replication stress.

This study reports an unusual ploidy-specific response to replication stress presented by a defective minichromosome maintenance (MCM) helicase allele in yeast. The corresponding mouse allele, Mcm4(Chaos3), predisposes mice to mammary gland tumors. While mcm4(Chaos3) causes replication stress in both haploid and diploid yeast, only diploid mutants exhibit G2/M delay, severe genetic instability (GIN), and reduced viability. These different outcomes are associated with distinct repair pathways adopted in haploid and diploid mutants. Haploid mutants use the Rad6-dependent pathways that resume stalled forks, whereas the diploid mutants use the Rad52- and MRX-dependent pathways that repair double strand breaks. The repair pathway choice is irreversible and not regulated by the availability of repair enzymes. This ploidy effect is independent of mating type heterozygosity and not further enhanced by increasing ploidy. In summary, a defective MCM helicase causes GIN only in particular cell types. In response to replication stress, early events associated with ploidy dictate the repair pathway choice. This study uncovers a fundamental difference between haplophase and diplophase in the maintenance of genome integrity.

Alternative mechanisms for coordinating polymerase alpha and MCM helicase.

Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase alpha in the absence of their presumed linker, Mcm10. Mcm10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Polalpha. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Polalpha without restoring the stability of Mcm10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the mcm10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that Mcm10 plays two important roles as a linker of the MCM helicase and Polalpha at the elongating replication fork--first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.

Aneuploidy and improved growth are coincident but not causal in a yeast cancer model.

Cancer cells have acquired mutations that alter their growth. Aneuploidy that typify cancer cells are often assumed to contribute to the abnormal growth characteristics. Here we test the idea of a link between aneuploidy and mutations allowing improved growth, using Saccharomyces cerevisiae containing a mcm4 helicase allele that was shown to cause cancer in mice. Yeast bearing this mcm4 allele are prone to undergoing a "hypermutable phase" characterized by a changing karyotype, ultimately yielding progeny with improved growth properties. When such progeny are returned to a normal karyotype by mating, their improved growth remains. Genetic analysis shows their improved growth is due to mutations in just a few loci. In sum, the effects of the mcm4 allele in mice are recapitulated in yeast, and the aneuploidy is not required to maintain improved growth.

Mcm10 mediates the interaction between DNA replication and silencing machineries.

The connection between DNA replication and heterochromatic silencing in yeast has been a topic of investigation for >20 years. While early studies showed that silencing requires passage through S phase and implicated several DNA replication factors in silencing, later works showed that silent chromatin could form without DNA replication. In this study we show that members of the replicative helicase (Mcm3 and Mcm7) play a role in silencing and physically interact with the essential silencing factor, Sir2, even in the absence of DNA replication. Another replication factor, Mcm10, mediates the interaction between these replication and silencing proteins via a short C-terminal domain. Mutations in this region of Mcm10 disrupt the interaction between Sir2 and several of the Mcm2-7 proteins. While such mutations caused silencing defects, they did not cause DNA replication defects or affect the association of Sir2 with chromatin. Our findings suggest that Mcm10 is required for the coupling of the replication and silencing machineries to silence chromatin in a context outside of DNA replication beyond the recruitment and spreading of Sir2 on chromatin.

Computational detection of significant variation in binding affinity across two sets of sequences with application to the analysis of replication origins in yeast.

BACKGROUND: In analyzing the stability of DNA replication origins in Saccharomyces cerevisiae we faced the question whether one set of sequences is significantly enriched in the number and/or the quality of the matches of a particular position weight matrix relative to another set. RESULTS: We present SADMAMA, a computational solution to a address this problem. SADMAMA implements two types of statistical tests to answer this question: one type is based on simplified models, while the other relies on bootstrapping, and as such might be preferable to users who are averse to such models. The bootstrap approach incorporates a novel "site-protected" resampling procedure which solves a problem we identify with naive resampling. CONCLUSION: SADMAMA's utility is demonstrated here by offering a plausible explanation to the differential ARS activity observed in our previous mcm1-1 mutant experiments 1, by suggesting the relevance of multiple weak ACS matches to efficient replication origin function in Saccharomyces cerevisiae, and by suggesting an explanation to the observed negative effect FKH2 has on chromatin silencing 2. SADMAMA is available for download from http://www.cs.cornell.edu/~keich/.

A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice.

Mcm4 (minichromosome maintenance-deficient 4 homolog) encodes a subunit of the MCM2-7 complex (also known as MCM2-MCM7), the replication licensing factor and presumptive replicative helicase. Here, we report that the mouse chromosome instability mutation Chaos3 (chromosome aberrations occurring spontaneously 3), isolated in a forward genetic screen, is a viable allele of Mcm4. Mcm4(Chaos3) encodes a change in an evolutionarily invariant amino acid (F345I), producing an apparently destabilized MCM4. Saccharomyces cerevisiae strains that we engineered to contain a corresponding allele (resulting in an F391I change) showed a classical minichromosome loss phenotype. Whereas homozygosity for a disrupted Mcm4 allele (Mcm4(-)) caused preimplantation lethality, Mcm(Chaos3/-) embryos died late in gestation, indicating that Mcm4(Chaos3) is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4(Chaos3/Chaos3) females succumbed to mammary adenocarcinomas with a mean latency of 12 months. These findings suggest that hypomorphic alleles of the genes encoding the subunits of the MCM2-7 complex may increase breast cancer risk.

Genome-wide hierarchy of replication origin usage in Saccharomyces cerevisiae.

Replication origins in a genome are inherently different in their base sequence and in their response to temporal and cell cycle regulation signals for DNA replication. To investigate the chromosomal determinants that influence the efficiency of initiation of DNA replication genome-wide, we made use of a reverse strategy originally used for the isolation of replication initiation mutants in Saccharomyces cerevisiae. In yeast, replication origins isolated from chromosomes support the autonomous replication of plasmids. These replication origins, whether in the context of a chromosome or a plasmid, will initiate efficiently in wild-type cells but show a dramatically contrasted efficiency of activation in mutants defective in the early steps of replication initiation. Serial passages of a genomic library of autonomously replicating sequences (ARSs) in such a mutant allowed us to select for constitutively active ARSs. We found a hierarchy of preferential initiation of ARSs that correlates with local transcription patterns. This preferential usage is enhanced in mutants defective in the assembly of the prereplication complex (pre-RC) but not in mutants defective in the activation of the pre-RC. Our findings are consistent with an interference of local transcription with the assembly of the pre-RC at a majority of replication origins.

Mcm10 is required for the maintenance of transcriptional silencing in Saccharomyces cerevisiae.

Mcm10 is an essential protein that participates in both the initiation and the elongation of DNA replication. In this study we demonstrate a role for Mcm10 in the maintenance of heterochromatic silencing at telomeres and HM loci of budding yeast. Two mcm10 mutants drastically reduce silencing of both URA3 and ADE2 reporter genes integrated into these silent loci. When exposed to alpha-factor, mcm10 mutant cells display a "shmoo-cluster" phenotype associated with a defect in the maintenance of silencing. In addition, when combined with a defect in the establishment of silent chromatin, mcm10 mutants demonstrate a synergistic defect in HML silencing. Consistent with a direct silencing function, Mcm10p shows a two-hybrid interaction with Sir2p and Sir3p that is destroyed by the mcm10-1 mutation and dependent on the C-terminal 108 amino acids. Tethering GBD-MCM10 to a defective HMR-E silencer is not sufficient to restore silencing. Furthermore, mutations in MCM10 inhibit the ability of GBD-SIR3 to restore silencing when tethered to a defective HMR-E. Suppressor mutations in MCM2, which suppress the temperature sensitivity of mcm10-1, fail to overcome the mcm10-1 silencing defect, suggesting that MCM10's role in transcriptional silencing may be separate from its essential functions in DNA replication.

Dual functional regulators coordinate DNA replication and gene expression in proliferating cells.

Gene products for cell growth must meet the pace of DNA replication and vice versa during the cell division cycle, therefore coordination of DNA replication and gene expression is vital to proliferating cells. During development in multicellular organisms when rapid cell divisions must be accompanied by the expression of particular gene sets in differentiating tissues, this coordination is even more crucial. Undoubtedly, multiple strategies are used to ensure the coordination of gene expression and DNA replication. In this review, we focus on the strategy that uses dual functional factors to serve both the functions of replication initiator and transcription regulator. Classical examples are the dual functional replication initiator/transcription regulators, DnaA of E. coli and T antigen of SV40, which bind replication origins and regulate their own synthesis. Emerging examples in eukaryotes are the growth responsive transcription factor E2f, the MADS domain combinatorial transcription factor Mcm1, and a subunit of the MCM2-7 helicase, Mcm7.

Mcm1 promotes replication initiation by binding specific elements at replication origins.

Minichromosome maintenance protein 1 (Mcm1) is required for efficient replication of autonomously replicating sequence (ARS)-containing plasmids in yeast cells. Reduced DNA binding activity in the Mcm1-1 mutant protein (P97L) results in selective initiation of a subset of replication origins and causes instability of ARS-containing plasmids. This plasmid instability in the mcm1-1 mutant can be overcome for a subset of ARSs by the inclusion of flanking sequences. Previous work showed that Mcm1 binds sequences flanking the minimal functional domains of ARSs. Here, we dissected two conserved telomeric X ARSs, ARS120 (XARS6L) and ARS131a (XARS7R), that replicate with different efficiencies in the mcm1-1 mutant. We found that additional Mcm1 binding sites in the C domain of ARS120 that are missing in ARS131a are responsible for efficient replication of ARS120 in the mcm1-1 mutant. Mutating a conserved Mcm1 binding site in the C domain diminished replication efficiency in ARS120 in wild-type cells, and increasing the number of Mcm1 binding sites stimulated replication efficiency. Our results suggest that threshold occupancy of Mcm1 in the C domain of telomeric ARSs is required for efficient initiation. We propose that origin usage in Saccharomyces cerevisiae may be regulated by the occupancy of Mcm1 at replication origins.

Mcm10 and Cdc45 cooperate in origin activation in Saccharomyces cerevisiae.

Mcm10 has recently been found to play a crucial role in multiple steps of the DNA replication initiation process in eukaryotes. Here, we have examined the role of Mcm10 in assembling initiation factors at a well-characterized yeast replication origin, ARS1. We find that the pre-replication complex (pre-RC) components Cdc6 and Mcm7 associate with ARS1 in the mcm10-1 mutant, suggesting that establishment of the pre-RC is not compromised in this mutant. Association of Cdc45 with ARS1 is reduced in the mcm10-1 mutant, suggesting that Mcm10 is involved in recruiting Cdc45 to the pre-RC. We find that overexpression of either Mcm10-1 or Cdc45 suppresses the growth defect of mcm10-1, and that a physical interaction between Cdc45 and Mcm10 is disrupted in the mcm10-1 mutant. Our results show that interaction between the Mcm10 and Cdc45 proteins facilitates the recruitment of Cdc45 onto the ARS1 origin.

Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation.

Mcm10 is required for the initiation of DNA replication in Saccharomyces cerevisiae. We have cloned MCM10 from Drosophila melanogaster and show that it complements a ScMCM10 null mutant. Moreover, Mcm10 interacts with key members of the prereplication complex: Mcm2, Dup (Cdt1), and Orc2. Interactions were also detected between Mcm10 and itself, Cdc45, and Hp1. RNAi depletion of Orc2 and Mcm10 in KC cells results in loss of DNA content. Furthermore, depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2, respectively, results in aberrant chromosome condensation. The condensation defects observed resemble previously published reports for Orc2, Orc5, and Mcm4 mutants. Our results strengthen and extend the argument that the processes of chromatin condensation and DNA replication are linked.

Evidence for a role of MCM (mini-chromosome maintenance)5 in transcriptional repression of sub-telomeric and Ty-proximal genes in Saccharomyces cerevisiae.

The MCM (mini-chromosome maintenance) genes have a well established role in the initiation of DNA replication and in the elongation of replication forks in Saccharomyces cerevisiae. In this study we demonstrate elevated expression of sub-telomeric and Ty retrotransposon-proximal genes in two mcm5 strains. This pattern of up-regulated genes resembles the genome-wide association of MCM proteins to chromatin that was reported earlier. We link the altered gene expression in mcm5 strains to a reversal of telomere position effect (TPE) and to remodeling of sub-telomeric and Ty chromatin. We also show a suppression of the Ts phenotype of a mcm5 strain by the high copy expression of the TRA1 component of the chromatin-remodeling SAGA/ADA (SPT-ADA-GCN5 acetylase/ADAptor). We propose that MCM proteins mediate the establishment of silent chromatin domains around telomeres and Ty retrotransposons.

Mcm7, a subunit of the presumptive MCM helicase, modulates its own expression in conjunction with Mcm1.

The Saccharomyces cerevisiae Mcm7 protein is a subunit of the presumed heteromeric MCM helicase that melts origin DNA and unwinds replication forks. Previous work showed that Mcm1 binds constitutively to the MCM7 promoter and regulates MCM7 expression. Here, we identify Mcm7 as a novel cofactor of Mcm1 in the regulation of MCM7 expression. Transcription of MCM7 is increased in the mcm7-1 mutant and decreased in the mcm1-1 mutant, suggesting that Mcm7 modulates its own expression in conjunction with Mcm1. Indeed, Mcm7 stimulates Mcm1 binding to the early cell cycle box upstream of the promoters of MCM7 as well as CDC6 and MCM5. Whereas Mcm1 binds these promoters constitutively, Mcm7 is recruited during late M phase, consistent with Mcm7 playing a direct role in modulating the periodic expression of early cell cycle genes. The multiple roles of Mcm7 in replication initiation, replication elongation, and autoregulation parallel those of the oncoprotein, the large T-antigen of the SV40 virus.

Budding yeast mcm10/dna43 mutant requires a novel repair pathway for viability.

BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Mcm10p functionally interacts with components of the pre-replicative complex (Mcm2-Mcm7 complex and origin recognition complex) as well as the pre-initiation complex component (Cdc45p) suggesting that it may be a component of the pre-RC as well as the pre-IC. Two-dimensional gel electrophoresis analysis showed that Mcm10p is required not only for the initiation of DNA synthesis at replication origins but also for the smooth passage of replication forks at origins. Genetic analysis showed that MCM10 interacts with components of the elongation machinery such as Pol delta and Pol epsilon, suggesting that it may play a role in elongation replication. RESULTS: We show that the mcm10 mutation causes replication fork pausing not only at potentially active origins but also at silent origins. We screened for mutations that are lethal in combination with mcm10-1 and obtained seven mutants named slm1-slm6 for synthetically lethal with mcm10. These mutants comprised six complementation groups that can be divided into three classes. Class 1 includes genes that encode components of the pre-RC and pre-IC and are represented by SLM3, 4 and 5 which are allelic to MCM7, MCM2 and CDC45, respectively. Class 2 includes genes involved in the processing of Okazaki fragments in lagging strand synthesis and is represented by SLM1, which is allelic to DNA2. Class 3 includes novel DNA repair genes represented by SLM2 and SLM6. CONCLUSIONS: The viability of the mcm10-1 mutant is dependent on a novel repair pathway that may participate either in resolving accumulated replication intermediates or the damage caused by blocked replication forks. These results are consistent with the hypothesis that Mcm10p is required for the passage of replication forks through obstacles such as those created by pre-RCs assembled at active or inactive replication origins.

Mcm1 binds replication origins.

Mcm1 is an essential protein required for the efficient replication of minichromosomes and the transcriptional regulation of early cell cycle genes in Saccharomyces cerevisiae. In this study, we report that Mcm1 is an abundant protein that associates globally with chromatin in a punctate pattern. We show that Mcm1 is localized at replication origins and plays an important role in the initiation of DNA synthesis at a chromosomal replication origin in vivo. Using purified Mcm1 protein, we show that Mcm1 binds cooperatively to multiple sites at autonomously replicating sequences. These results suggest that, in addition to its role as a transcription factor for the expression of replication genes, Mcm1 may influence the local structure of replication origins by direct binding.

Mcm3 is polyubiquitinated during mitosis before establishment of the pre-replication complex.

To ensure fidelity in genome duplication, eukaryotes restrict DNA synthesis to once every cell division by a cascade of regulated steps. Central to this cascade is the periodic assembly of the hexameric MCM2-7 complex at replication origins. However, in Saccharomyces cerevisiae, only a fraction of each MCM protein is able to assemble into hexamers and associate with replication origins during M phase, suggesting that MCM complex assembly and recruitment may be regulated post-translationally. Here we show that a small fraction of Mcm3p is polyubiquitinated at the onset of MCM complex assembly. Reducing the rate of ubiquitination by uba1-165, a suppressor of mcm3-10, restored the interaction of Mcm3-10p with subunits of the MCM complex and its recruitment to the replication origin. Possible roles for ubiquitinated Mcm3p in the assembly of the MCM complex at replication origins are discussed.

The Methanobacterium thermoautotrophicum MCM protein can form heptameric rings.

Mini-chromosome maintenance (MCM) proteins form a conserved family found in all eukaryotes and are essential for DNA replication. They exist as heteromultimeric complexes containing as many as six different proteins. These complexes are believed to be the replicative helicases, functioning as hexameric rings at replication forks. In most archaea a single MCM protein exists. The protein from Methanobacterium thermoautotrophicum (mtMCM) has been reported to assemble into a large complex consistent with a dodecamer. We show that mtMCM can assemble into a heptameric ring. This ring contains a C-terminal helicase domain that can be fit with crystal structures of ring helicases and an N-terminal domain of unknown function. While the structure of the ring is very similar to that of hexameric replicative helicases such as bacteriophage T7 gp4, our results show that such ring structures may not be constrained to have only six subunits.

Two mcm3 mutations affect different steps in the initiation of DNA replication.

Mcm3 is a subunit of the hexameric MCM2-7 complex required for the initiation and elongation of DNA replication in eukaryotes. We have characterized two mutant alleles, mcm3-1 and mcm3-10, in Saccharomyces cerevisiae and showed that they are defective at different steps of the replication initiation process. Mcm3-10 contains a P118L substitution that compromises its interaction with Mcm5 and the recruitment of Mcm3 and Mcm7 to a replication origin. P118 is conserved between Mcm3, Mcm4, Mcm5, and Mcm7. An identical substitution of this conserved residue in Mcm5 (P83L of mcm5-bob1) strengthens the interaction between Mcm3 and Mcm5 and allows cells to enter S phase independent of Cdc7-Dbf4 kinase (Hardy, C. F., Dryga, O., Pahl, P. M. B., and Sclafani, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3151-3155). Mcm3-1 contains a G246E mutation that diminishes the efficiency of replication initiation (Yan, H., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160) but not its interaction with Mcm5 or recruitment of the MCM2-7 complex to replication origin. These observations indicate that Mcm3-10 is defective in a step before, and Mcm3-1 is defective in a step after the recruitment of the MCM2-7 complex to replication origins.