The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.     

Minichromosome maintenance as a genetic assay for defects in DNA replication.

Minichromosome maintenance (mcm) is an effective genetic assay for mutants defective in DNA replication. Two classes of mcm mutants have been identified using this screen: those that differentially affect the activities of certain autonomously replicating sequences (ARSs) and those that uniformly affect the activities of all ARSs. The ARS-specific MCM genes are essential for the initiation of DNA replication. Among these are members of the MCM2-7 family that encode subunits of the preinitiation complex and MCM10, whose gene product interacts with members of the Mcm2-7 proteins. Among the ARS-nonspecific MCM gene products are chromosome transmission factors. Refinement of this genetic assay as a screening tool and further analysis of existing mcm mutants may reveal new replication initiation proteins.     

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.     

Essential role of MCM proteins in premeiotic DNA replication

A critical event in eukaryotic DNA replication involves association of minichromosome maintenance (MCM2-7) proteins with origins, to form prereplicative complexes (pre-RCs) that are competent for initiation. The ability of mutants defective in MCM2-7 function to complete meiosis had suggested that pre-RC components could be irrelevant to premeiotic S phase. We show here that MCM2-7 proteins bind to chromatin in fission yeast cells preparing for meiosis and during premeiotic S phase in a manner suggesting they in fact are required for DNA replication in the meiotic cycle. This is confirmed by analysis of a degron mcm4 mutant, which cannot carry out premeiotic DNA replication. Later in meiosis, Mcm4 chromatin association is blocked between meiotic nuclear divisions, presumably accounting for the absence of a second round of DNA replication. Together, these results emphasize similarity between replication mechanisms in mitotic and meiotic cell cycles.     

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.     

Functional conservation of beta-hairpin DNA binding domains in the Mcm protein of Methanobacterium thermoautotrophicum and the Mcm5 protein of Saccharomyces cerevisiae

Mcm proteins are an important family of evolutionarily conserved helicases required for DNA replication in eukaryotes. The eukaryotic Mcm complex consists of six paralogs that form a heterohexameric ring. Because the intact Mcm2-7 hexamer is inactive in vitro, it has been difficult to determine the precise function of the different subunits. The solved atomic structure of an archaeal minichromosome maintenance (MCM) homolog provides insight into the function of eukaryotic Mcm proteins. The N-terminal positively charged central channel in the archaeal molecule consists of beta-hairpin domains essential for DNA binding in vitro. Eukaryotic Mcm proteins also have beta-hairpin domains, but their function is unknown. With the archaeal atomic structure as a guide, yeast molecular genetics was used to query the function of the beta-hairpin domains in vivo. A yeast mcm5 mutant with beta-hairpin mutations displays defects in the G1/S transition of the cell cycle, the initiation phase of DNA replication, and in the binding of the entire Mcm2-7 complex to replication origins. A similar mcm4 mutation is synthetically lethal with the mcm5 mutation. Therefore, in addition to its known regulatory role, Mcm5 protein has a positive role in origin binding, which requires coordination by all six Mcm2-7 subunits in the hexamer.     

Structural changes in Mcm5 protein bypass Cdc7-Dbf4 function and reduce replication origin efficiency in Saccharomyces cerevisiae

Eukaryotic chromosomal replication is a complicated process with many origins firing at different efficiencies and times during S phase. Prereplication complexes are assembled on all origins in G(1) phase, and yet only a subset of complexes is activated during S phase by DDK (for Dbf4-dependent kinase) (Cdc7-Dbf4). The yeast mcm5-bob1 (P83L) mutation bypasses DDK but results in reduced intrinsic firing efficiency at 11 endogenous origins and at origins located on minichromosomes. Origin efficiency may result from Mcm5 protein assuming an altered conformation, as predicted from the atomic structure of an archaeal MCM (for minichromosome maintenance) homologue. Similarly, an intragenic mutation in a residue predicted to interact with P83L suppresses the mcm5-bob1 bypass phenotype. We propose DDK phosphorylation of the MCM complex normally results in a single, highly active conformation of Mcm5, whereas the mcm5-bob1 mutation produces a number of conformations, only one of which is permissive for origin activation. Random adoption of these alternate states by the mcm5-bob1 protein can explain both how origin firing occurs independently of DDK and why origin efficiency is reduced. Because similar mutations in mcm2 and mcm4 cannot bypass DDK, Mcm5 protein may be a unique Mcm protein that is the final target of DDK regulation.     

CDC45, a novel yeast gene that functions with the origin recognition complex and Mcm proteins in initiation of DNA replication.

The CDC45 gene of Saccharomyces cerevisiae was isolated by complementation of the cold-sensitive cdc45-1 mutant and shown to be essential for cell viability. Although CDC45 genetically interacts with a group of MCM genes (CDC46, CDC47, and CDC54), the predicted sequence of its protein product reveals no significant sequence similarity to any known Mcm family member. Further genetic characterization of the cdc45-1 mutant demonstrated that it is synthetically lethal with orc2-1, mcm2-1, and mcm3-1. These results not only reveal a functional connection between the origin recognition complex (ORC) and Cdc45p but also extend the CDC45-MCM genetic interaction to all known MCM family members that were shown to be involved in replication initiation. Initiation of DNA replication in cdc45-1 cells was defective, causing a delayed entry into S phase at the nonpermissive temperature, as well as a high plasmid loss rate which could be suppressed by tandem copies of replication origins. Furthermore, two-dimensional gels directly showed that chromosomal origins fired less frequently in cdc45-1 cells at the nonpermissive temperature. These findings suggest that Cdc45p, ORC, and Mcm proteins act in concert for replication initiation throughout the genome.     

Overexpression of a novel gene , Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor

INTRODUCTIONDNA replication is a fundamenial process thatmust occur only once at each ce1l cycle. This restrictcontrol appears to be achieved through the coordi-nated actiVities of numerous proteins. The buddingyeast Saccharompes cerevhaae provides an excellenteukaryotic model fOr study of proteins invo1ved inthe control of DNA replication.In the budding yeast, minichromosome mainte-nance (MCM) proteins, MCM2-7, are a family of strsequence-related proteins that play crucia1 roles inr…     

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.     

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.     

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.     

Overexpression of a novel gene, Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor.

MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutant (mcm10-1) of budding yeast shows a growth arrest at 37 degrees C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 degrees C. Interestingly, this mcm10-1 suppressor undergoes cell cycle arrest at 14 degrees C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of transformation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 degrees C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.     

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.