Equilibrium between nascent and parental MCM proteins protects replicating genomes

Abstract The MAD2-dependent spindle checkpoint blocks anaphase until all chromosomes have achieved successful bipolar attachment to the mitotic spindle. The DNA damage and DNA replication checkpoints block anaphase in response to DNA lesions that may include single-stranded DNA and stalled replication forks. Many of the same conditions that activate the DNA damage and DNA replication checkpoints also activated the spindle checkpoint. The mad2Δ mutation partially relieved the arrest responses of cells to mutations affecting the replication proteins Mcm3p and Pol1p. Thus a previously unrecognized aspect of spindle checkpoint function may be to protect cells from defects in DNA replication. Furthermore, in cells lacking either the DNA damage or the DNA replication checkpoints, the spindle checkpoint contributed to the arrest responses of cells to the DNA-damaging agent methyl methanesulfonate, the replication inhibitor hydroxyurea, and mutations affecting Mcm2p and Orc2p. Thus the spindle checkpoint was sensitive to a wider range of chromosomal perturbations than previously recognized. Finally, the DNA replication checkpoint did not contribute to the arrests of cells in response to mutations affecting ORC, Mcm proteins, or DNA polymerase δ. Thus the specificity of this checkpoint may be more limited than previously recognized.

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Role of DNA Replication Proteins in Double-Strand Break-Induced Recombination in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.6891-6899.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 6891-6899 ◽

Cited By ~ 92

Author(s):

Xuan Wang ◽

Grzegorz Ira ◽

José Antonio Tercero ◽

Allyson M. Holmes ◽

John F. X. Diffley ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Synthesis ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

S Phase ◽

Temperature Sensitive ◽

Replication Proteins ◽

Dna Ligases ◽

Mcm Proteins

ABSTRACT Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4Δ strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase α (Polα), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G2-arrested cells. Whereas PCNA was still essential for MAT switching, neither Polα nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Polα-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

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The role of MCM proteins in the cell cycle control of genome duplication

BioEssays ◽

10.1002/bies.950180305 ◽

1996 ◽

Vol 18 (3) ◽

pp. 183-190 ◽

Cited By ~ 86

Author(s):

Stephen E. Kearsey ◽

Domenico Maiorano ◽

Eddie C. Holmes ◽

Ivan T. Todorov

Keyword(s):

Cell Cycle ◽

Genome Duplication ◽

Cell Cycle Control ◽

Mcm Proteins

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DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

Journal of Biological Chemistry ◽

10.1074/jbc.m407772200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45586-45593 ◽

Cited By ~ 30

Author(s):

David Shechter ◽

Carol Y. Ying ◽

Jean Gautier

Keyword(s):

Dna Replication ◽

Neutralizing Antibodies ◽

Atp Hydrolysis ◽

Initial Period ◽

Dna Helicase ◽

Dna Unwinding ◽

Origin Firing ◽

Replicative Helicase ◽

Mcm Proteins

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

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Drosophila Minichromosome Maintenance 6 Is Required for Chorion Gene Amplification and Genomic Replication

Molecular Biology of the Cell ◽

10.1091/mbc.01-08-0400 ◽

2002 ◽

Vol 13 (2) ◽

pp. 607-620 ◽

Cited By ~ 48

Author(s):

Gina Schwed ◽

Noah May ◽

Yana Pechersky ◽

Brian R. Calvi

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Genetic Dissection ◽

Minichromosome Maintenance ◽

Multiple Origins ◽

Origin Licensing ◽

Mcm Complex ◽

Mcm Proteins ◽

Prereplicative Complex ◽

Cycle Regulation

Duplication of the eukaryotic genome initiates from multiple origins of DNA replication whose activity is coordinated with the cell cycle. We have been studying the origins of DNA replication that control amplification of eggshell (chorion) genes duringDrosophila oogenesis. Mutation of genes required for amplification results in a thin eggshell phenotype, allowing a genetic dissection of origin regulation. Herein, we show that one mutation corresponds to a subunit of the minichromosome maintenance (MCM) complex of proteins, MCM6. The binding of the MCM complex to origins in G1 as part of a prereplicative complex is critical for the cell cycle regulation of origin licensing. We find that MCM6 associates with other MCM subunits during amplification. These results suggest that chorion origins are bound by an amplification complex that contains MCM proteins and therefore resembles the prereplicative complex. Lethal alleles of MCM6 reveal it is essential for mitotic cycles and endocycles, and suggest that its function is mediated by ATP. We discuss the implications of these findings for the role of MCMs in the coordination of DNA replication during the cell cycle.

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Faculty Opinions recommendation of A rotary pumping model for helicase function of MCM proteins at a distance from replication forks.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1011757.193102 ◽

2003 ◽

Author(s):

Peter B Becker

Keyword(s):

Replication Forks ◽

Mcm Proteins

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Mammalian nuclei become licensed for DNA replication during late telophase

Journal of Cell Science ◽

10.1242/jcs.115.1.51 ◽

2002 ◽

Vol 115 (1) ◽

pp. 51-59 ◽

Cited By ~ 4

Author(s):

Daniela S. Dimitrova ◽

Tatyana A. Prokhorova ◽

J. Julian Blow ◽

Ivan T. Todorov ◽

David M. Gilbert

Keyword(s):

Cho Cells ◽

Chinese Hamster ◽

Significant Degree ◽

Initiation Of Replication ◽

Egg Extracts ◽

Late Telophase ◽

Xenopus Egg ◽

Mcm Proteins ◽

Nuclear Envelope Formation ◽

Xenopus Egg Extracts

Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.

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Expression, phosphorylation and nuclear localization of the human P1 protein, a homologue of the yeast Mcm 3 replication protein

Journal of Cell Science ◽

10.1242/jcs.108.4.1381 ◽

1995 ◽

Vol 108 (4) ◽

pp. 1381-1389 ◽

Cited By ~ 2

Author(s):

D. Schulte ◽

R. Burkhart ◽

C. Musahl ◽

B. Hu ◽

C. Schlatterer ◽

...

Keyword(s):

Sequence Similarity ◽

Protein A ◽

Fold Increase ◽

S Phase ◽

Human Protein ◽

Replication Protein ◽

High Sequence Similarity ◽

Central Regions ◽

Mcm Proteins ◽

Carboxy Terminal

The human protein P1 belongs to a newly discovered class of mammalian nuclear proteins with high sequence homology to yeast replication proteins. We present the entire amino acid sequence of the human protein P1 as predicted from the cDNA sequence, and show that P1 shares three central regions of high sequence similarity (about 75%) and a highly hydrophilic carboxy-terminal region with the yeast Mcm3 replication protein. The human genome most probably contains one P1 gene which is activated when HeLa cells progress to S phase, as shown by a several-fold increase in P1-specific mRNA. However, the amounts of P1 protein do not detectably change during this period, but P1 protein becomes phosphorylated at the beginning of S phase. In contrast to the yeast Mcm proteins, which disappear from nuclei after initiation of DNA replication, protein P1 remains in the nucleus during and after S phase. P1 is dispersed in mitotic cells and may be excluded from binding to chromosomes.

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