scholarly journals Minichromosome Maintenance Proteins Interact with Checkpoint and Recombination Proteins To Promote S-Phase Genome Stability

2008 ◽  
Vol 28 (5) ◽  
pp. 1724-1738 ◽  
Author(s):  
Julie M. Bailis ◽  
Douglas D. Luche ◽  
Tony Hunter ◽  
Susan L. Forsburg
Keyword(s):  
Dna Synthesis ◽  
Replication Fork ◽  
Genome Stability ◽  
S Phase ◽  
Minichromosome Maintenance ◽  
Replication Checkpoint ◽  
Recombination Proteins ◽  
Mcm Complex ◽  
Mcm Proteins ◽  
The Stability

ABSTRACT The minichromosome maintenance (MCM) complex plays essential, conserved roles throughout DNA synthesis: first, as a component of the prereplication complex at origins and, then, as a helicase associated with replication forks. Here we use fission yeast (Schizosaccharomyces pombe) as a model to demonstrate a role for the MCM complex in protecting replication fork structure and promoting recovery from replication arrest. Loss of MCM function generates lethal double-strand breaks at sites of DNA synthesis during replication elongation, suggesting replication fork collapse. MCM function also maintains the stability of forks stalled by hydroxyurea that activate the replication checkpoint. In cells where the checkpoint is activated, Mcm4 binds the Cds1 kinase and undergoes Cds1-dependent phosphorylation. MCM proteins also interact with proteins involved in homologous recombination, which promotes recovery from arrest by ensuring normal mitosis. We suggest that the MCM complex links replication fork stabilization with checkpoint arrest and recovery through direct interactions with checkpoint and recombination proteins and that this role in S-phase genome stability is conserved from yeast to human cells.

scholarly journals Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication

2018 ◽  
Vol 29 (25) ◽  
pp. 2989-3002 ◽  
Author(s):  
Pedro N. Pozo ◽  
Jacob P. Matson ◽  
Yasemin Cole ◽  
Katarzyna M. Kedziora ◽  
Gavin D. Grant ◽  
...  
Keyword(s):  
Genome Stability ◽  
Homology Model ◽  
Cyclin A ◽  
Genome Replication ◽  
Minichromosome Maintenance ◽  
Interaction Domain ◽  
Origin Licensing ◽  
Primordial Dwarfism ◽  
Mcm Complex ◽  
The Stability

The earliest step in DNA replication is origin licensing, which is the DNA loading of minichromosome maintenance (MCM) helicase complexes. The Cdc10-dependent transcript 1 (Cdt1) protein is essential for MCM loading during the G1 phase of the cell cycle, but the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (the Meier–Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than wild-type (WT) Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1–MCM complex positions the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele ( Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live-cell imaging analysis demonstrated no change in the stability of this variant, however. Instead, we propose that cyclin A/CDK inhibits the Cdt1 licensing function independent of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.

scholarly journals The eukaryotic replisome requires an additional helicase to disarm dormant replication origins

2020 ◽  
Author(s):  
Jake Hill ◽  
Patrik Eickhoff ◽  
Lucy S. Drury ◽  
Alessandro Costa ◽  
John F.X. Diffley
Keyword(s):  
Replication Fork ◽  
S Phase ◽  
Replication Forks ◽  
Minichromosome Maintenance ◽  
Double Stranded Dna ◽  
Efficient Replication ◽  
Eukaryotic Dna ◽  
Mcm Proteins ◽  
Fork Stalling ◽  
Pif1 Helicase

Origins of eukaryotic DNA replication are ‘licensed’ during G1 phase of the cell cycle by loading the six related minichromosome maintenance (MCM) proteins into a double hexameric ring around double-stranded DNA. In S phase, some double hexamers (MCM DHs) are converted into active CMG (Cdc45-MCM-GINS) helicases which nucleate assembly of bidirectional replication forks. The remaining unfired MCM DHs act as ‘dormant’ origins to provide backup replisomes in the event of replication fork stalling. The fate of unfired MCM DHs during replication is unknown. Here we show that active replisomes cannot remove unfired MCM DHs. Instead, they are pushed ahead of the replisome where they prevent fork convergence during replication termination and replisome progression through nucleosomes. Pif1 helicase, together with the replisome, can remove unfired MCM DHs specifically from replicating DNA, allowing efficient replication and termination. Our results provide an explanation for how excess replication license is removed during S phase.

scholarly journals The eukaryotic replisome requires an additional helicase to disarm dormant replication origins

2020 ◽  
Author(s):  
Jake Hill ◽  
Patrik Eickhoff ◽  
Lucy Drury ◽  
Alessandro Costa ◽  
John Diffley
Keyword(s):  
Replication Fork ◽  
S Phase ◽  
Replication Forks ◽  
Minichromosome Maintenance ◽  
Double Stranded Dna ◽  
Efficient Replication ◽  
Eukaryotic Dna ◽  
Mcm Proteins ◽  
Fork Stalling ◽  
Pif1 Helicase

Abstract Origins of eukaryotic DNA replication are ‘licensed’ during G1 phase of the cell cycle by loading the six related minichromosome maintenance (MCM) proteins into a double hexameric ring around double-stranded DNA. In S phase, some double hexamers (MCM DHs) are converted into active CMG (Cdc45-MCM-GINS) helicases which nucleate assembly of bidirectional replication forks. The remaining unfired MCM DHs act as ‘dormant’ origins to provide backup replisomes in the event of replication fork stalling. The fate of unfired MCM DHs during replication is unknown. Here we show that active replisomes cannot remove unfired MCM DHs. Instead, they are pushed ahead of the replisome where they prevent fork convergence during replication termination and replisome progression through nucleosomes. Pif1 helicase, together with the replisome, can remove unfired MCM DHs specifically from replicating DNA, allowing efficient replication and termination. Our results provide an explanation for how excess replication license is removed during S phase.

scholarly journals Nascent Transcription of MCM2-7 Is Important for Nuclear Localization of the Minichromosome Maintenance Complex in G1

2007 ◽  
Vol 18 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Katherine A. Braun ◽  
Linda L. Breeden
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Minichromosome Maintenance ◽  
Mcm Complex ◽  
Mcm Proteins ◽  
Constitutive Production ◽  
New Protein

The minichromosome maintenance genes (MCM2-7) are transcribed at M/G1 just as the Mcm complex is imported into the nucleus to be assembled into prereplication complexes, during a period of low cyclin-dependent kinase (CDK) activity. The CDKs trigger DNA replication and prevent rereplication in part by exporting Mcm2-7 from the nucleus during S phase. We have found that repression of MCM2-7 transcription in a single cell cycle interferes with the nuclear import of Mcms in the subsequent M/G1 phase. This suggests that nascent Mcm proteins are preferentially imported into the nucleus. Consistent with this, we find that loss of CDK activity in G2/M is not sufficient for nuclear import, there is also a requirement for new protein synthesis. This requirement is not met by constitutive production of Cdc6 and does not involve synthesis of new transport machinery. The Mcm proteins generated in the previous cell cycle, which are unable to reaccumulate in the nucleus, are predominantly turned over by ubiquitin-mediated proteolysis in late mitosis/early G1. Therefore, the nuclear localization of Mcm2-7 is dependent on nascent transcription and translation of Mcm2-7 and the elimination of CDK activity which occurs simultaneously as cells enter G1.

scholarly journals Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

2011 ◽  
Vol 192 (1) ◽  
pp. 29-41 ◽  
Author(s):  
Marjorie A. Kuipers ◽  
Timothy J. Stasevich ◽  
Takayo Sasaki ◽  
Korey A. Wilson ◽  
Kristin L. Hazelwood ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Replication Fork ◽  
S Phase ◽  
G1 Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycles ◽  
Replication Arrest ◽  
Minichromosome Maintenance Protein ◽  
Mcm Proteins

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.

scholarly journals A catalytic-independent function of human DNA polymerase Kappa controls the pool of the Checkpoint Kinase 1

2021 ◽  
Author(s):  
Marina Dall’Osto ◽  
Laura Pierini ◽  
Nicolas Valery ◽  
Jean-Sébastien Hoffmann ◽  
Marie-jeanne Pillaire
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Genome Stability ◽  
Replication Process ◽  
Replication Checkpoint ◽  
Protein Pool ◽  
Synthesis Activity ◽  
The Stability ◽  
Y Family ◽  
Major Mediator

ABSTRACTDNA polymerase kappa (Pol κ) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate and replication checkpoint at stalled forks.Here we unveiled an unexpected role for Pol κ in controlling the stability and abundance of Chk1, the major mediator of the replication checkpoint. We found that loss of Pol κ decreased the Chk1 protein level in the nucleus of four human cell lines. Pol κ and not the other Y‐family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol κ depletion affected the protein stability of Chk1 and protected it from proteasome degradation and the replication recovery defects observed in Pol κ-depleted cells could be overcome by the re-expression of Chk1. Importantly, this new function of Pol κ does not require its catalytic activity, revealing that in addition to its known roles in the replication process, Pol κ can contribute to the maintenance of genome stability independently of its DNA synthesis activity.

The MCM helicase: linking checkpoints to the replication fork

2008 ◽  
Vol 36 (1) ◽  
pp. 114-119 ◽  
Author(s):  
Susan L. Forsburg
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Integrity ◽  
Minichromosome Maintenance ◽  
Replication Checkpoint ◽  
Checkpoint Kinases ◽  
Mcm Helicase ◽  
Mcm Proteins

The MCM (minichromosome maintenance) complex is a helicase which is essential for DNA replication. Recent results suggest that the MCM helicase is important for replication fork integrity, and may function as a target of the replication checkpoint. Interactions between MCM proteins, checkpoint kinases, and repair and recovery proteins suggest that MCMs are proximal effectors of replication fork stability in the cell and are likely to play an important role in maintaining genome integrity.

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

2004 ◽  
Vol 24 (16) ◽  
pp. 6891-6899 ◽  
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.

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

2002 ◽  
Vol 13 (2) ◽  
pp. 607-620 ◽  
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.

scholarly journals Structural Changes in Mcm5 Protein Bypass Cdc7-Dbf4 Function and Reduce Replication Origin Efficiency in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (21) ◽  
pp. 7594-7602 ◽  
Author(s):  
Margaret L. Hoang ◽  
Ronald P. Leon ◽  
Luis Pessoa-Brandao ◽  
Sonia Hunt ◽  
M. K. Raghuraman ◽  
...  
Keyword(s):  
Structural Changes ◽  
S Phase ◽  
Minichromosome Maintenance ◽  
Chromosomal Replication ◽  
Origin Firing ◽  
Highly Active ◽  
Mcm Complex ◽  
Alternate States ◽  
Firing Efficiency ◽  
Intrinsic Firing

ABSTRACT 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 G1 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.

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