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PubMed2004

Structure-forming CAG/CTG repeat sequences are sensitive to breakage in the absence of Mrc1 checkpoint function and S-phase checkpoint signaling: implications for trinucleotide repeat expansion diseases.

Expansion of trinucleotide repeat sequences is the cause of multiple inherited human genetic diseases including Huntington's disease and myotonic dystrophy. CTG and CAG repeats have been shown to form stable secondary structures that can impair Okazaki fragment processing and may impede replication fork progression. We recently showed that mutation of DNA damage checkpoint proteins results in increased chromosome breaks at expanded CAG/CTG repeats and in increased repeat instability (expansions and contractions).(1) Here we report that long CAG approximately 155 tracts are especially sensitive to the absence of Mrc1 (Claspin) checkpoint function, implicating the S-phase checkpoint in maintenance of trinucleotide repeats and other secondary-structure forming sequences. Based on all of our results, we propose a model for the detection of different types of structures by different checkpoint signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Maintenance DNA methylation of nucleosome core particles.

The enzyme responsible for maintenance methylation of CpG dinucleotides in vertebrates is DNMT1. The presence of DNMT1 in DNA replication foci raises the issue of whether this enzyme needs to gain access to nascent DNA before its packaging into nucleosomes, which occurs very rapidly behind the replication fork. Using nucleosomes positioned along the 5 S rRNA gene, we find that DNMT1 is able to methylate a number of CpG sites even when the DNA major groove is oriented toward the histone surface. However, we also find that the ability of DNMT1 to methylate nucleosomal sites is highly dependent on the nature of the DNA substrate. Although nucleosomes containing the Air promoter are refractory to methylation irrespective of target cytosine location, nucleosomes reconstituted onto the H19 imprinting control region are more accessible. These results argue that although DNMT1 is intrinsically capable of methylating some DNA sequences even after their packaging into nucleosomes, this is not the case for at least a fraction of DNA sequences whose function is regulated by DNA methylation.

Animals↗

PubMed2002

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.

Amino Acid Motifs↗

PubMed2006

Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases.

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.

Amino Acid Sequence↗

PubMed2002

Checking on the fork: the DNA-replication stress-response pathway.

To ensure the fidelity of DNA replication, cells activate a stress-response pathway when DNA replication is perturbed. This pathway regulates not only progress through the cell cycle but also transcription, apoptosis, DNA repair/recombination and DNA replication itself. Mounting evidence has suggested that this pathway is important for the maintenance of genomic integrity. Here, we discuss recent findings about how this pathway is activated by replication stress and how it regulates the DNA-replication machinery to alleviate the stress.

Animals↗

PubMed2004

DNA unwinding is an Mcm complex-dependent and ATP hydrolysis-dependent process.

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 activities in 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) from Xenopus laevis eggs and six purified polyclonal antibodies generated against each of the Xenopus Mcm 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 p21(cip1) specifically blocks origin firing but does not prevent helicase action. When p21(cip1) is added, followed by the non-hydrolyzable analog ATPgammaS 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.

Adenosine Triphosphate↗

PubMed1990

Evidence that replication initiates at only some of the potential origins in each oligomeric form of bovine papillomavirus type 1 DNA.

In a subclone of ID13 mouse fibroblasts latently infected with bovine papillomavirus type 1 (BPV-1) DNA, the viral genome occurred as a mixture of extrachromosomal circular monomers and oligomers. Multiple copies were also associated with the host cell genome, predominantly at a single site in a head-to-tail tandem array. We examined the replicative intermediates of extrachromosomal forms of BPV-1 DNA by using two-dimensional gel electrophoresis. The results obtained indicate that initiation of DNA replication occurred near the center of the EcoRI-BamHI 5.6-kilobase fragment. In some molecules, however, this fragment was replicated from one end to the other by means of a single fork initiated elsewhere. Termination also occurred within this fragment. The EcoRI-BamHI 2.3-kilobase fragment replicated as a DNA molecule containing a termination site for DNA replication and also by means of a single fork traversing the fragment from one end to the other. Thus, replication forks proceeded through these fragments in different manners, apparently depending on whether they were part of a monomer, a dimer, a trimer, or higher oligomers. These observations lead to the conclusion that initiation of DNA replication in BPV-1 DNA takes place at or close to plasmid maintenance sequence 1. From this point, replication proceeds bidirectionally and termination occurs approximately 180 degrees opposite the origin. The results obtained are consistent with one or more replication origins being quiescent in BPV-1 DNA oligomers.

Animals↗

PubMed2014

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.

Cell Cycle Proteins↗

PubMed2006

Role of Claspin in regulation of nucleotide excision repair factor DDB2.

The replication checkpoint protein Claspin is important for maintenance of genomic stability and is required for cells to overcome genotoxic stress. Upon UV-induced DNA damage, Claspin is required for activation of the ATR-mediated DNA damage checkpoint response, leading to arrest of DNA replication and inhibition of cell cycle progression. Located at the DNA replication fork, Claspin is also suggested to monitor replication and sense damage. Our present studies in HeLa cells demonstrate associations between the Claspin/ATR-related DNA damage checkpoint response and the global genomic nucleotide excision repair pathway. siRNA-mediated knockdown of Claspin abolishes the UV-induced degradation of DDB2 and impairs the co-localization of DDB2 to DNA damage sites. Thus, the presence of Claspin is required for the total turnover of DNA damage binding protein DDB2, as well as for its functionality in DNA damage recognition. Claspin, however, seems not to be required for maintaining the cellular level of the NER factor XPC and its UV-induced post-translational modifications. Co-localization of XPC with DNA lesions is also intact in the absence of Claspin as is the repair of the UV-induced lesions CPD and 6-4PP. Claspin itself may be directly responsible for physical interaction between the two pathways since Claspin is able to associate with DDB1, DDB2 and XPC. Taken together, these findings reveal physical and functional interplay between Claspin and NER-related proteins and demonstrate crosstalk between the DNA damage checkpoint control and DNA damage repair pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2006

RecQ helicases: lessons from model organisms.

RecQ DNA helicases function during DNA replication and are essential for the maintenance of genome stability. There is increasing evidence that spontaneous genomic instability occurs primarily during DNA replication, and that proteins involved in the S-phase checkpoint are a principal defence against such instability. Cells that lack functional RecQ helicases exhibit phenotypes consistent with an inability to fully resume replication fork progress after encountering DNA damage or fork arrest. In this review we will concentrate on the various functions of RecQ helicases during S phase in model organisms.

Adenosine Triphosphatases↗

PubMed2003

RecQ helicases: multiple roles in genome maintenance.

The RecQ helicases are highly conserved in evolution and are required for maintaining genome stability in all organisms. In humans, loss of RecQ helicase function is associated with predisposition to cancer and/or premature ageing. Recent data show that RecQ helicases have several roles during S phase of the cell cycle, ranging from facilitating the resumption of DNA synthesis at sites of replication fork breakdown to resolving structures during the process of homologous recombination.

Animals↗

PubMed1997

Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase.

In S. cerevisiae, the chromatin structure of DNA replication origins changes as cells become competent for DNA replication, suggesting that G1 phase-specific association of replication factors with origin DNA regulates entry into S phase. We demonstrate that ORC, Cdc45p, and MCM proteins are components of prereplication complexes (pre-RC). The MCM-origin association is dependent upon ORC and Cdc6p. During S phase, MCM proteins and Cdc45p dissociate from origin DNA and associate with nonorigin DNA with similar kinetics as DNA Polymerase epsilon, which is present at DNA replication forks. Our results identify protein components of the pre-RC and a novel replication complex appearing at the G1/S transition (the RC), and suggest that after initiation MCM proteins and Cdc45p move with eukaryotic replication forks.

Carrier Proteins↗

PubMed2026

Direct visualization of MCM helicase activation and replisome coupling in situ.

Deciphering the spatial organization of molecular machines that copy the genome remains a fundamental challenge in biology. Essential for eukaryotic DNA replication, Mini-Chromosome Maintenance (MCM2-7) helicases are loaded during G1 as double hexamers (DHs) to license replication origins. Upon activation in S phase, each DH is thought to split into two single hexamers (SHs) that form the active CMG helicases and travel bidirectionally. However, the field has long been divided: biochemical and structural studies define CMG helicases as autonomous, independent motors, while genomic and cellular imaging assays suggest sister replisomes remain physically coupled within replication factories. Here, we use MINFLUX nanoscopy to localize individual MCM complexes down to nanometer precision in situ, directly resolving DHs in human cells and capturing their separation into SHs upon origin firing. We find that the resulting sister replisomes do not diffuse apart: they remain coupled at a characteristic distance of ~40 nm throughout S phase. Depletion experiments identify two distinct contributions to this coupling: local, protein-mediated tethering by the AND1 scaffold, and higher-order spatial confinement dependent on cohesin, which is dispensable for MCM loading in G1 but required to maintain coupling in S phase. By linking the nanometer-scale architecture of the replisome to the genome-wide topology of replication fountains, these findings provide direct spatial evidence that sister forks are coupled during DNA synthesis and define the molecular forces that organize replisomes within their native nuclear context.

DNA replication↗

PubMed2002

RecQ helicases: at the heart of genetic stability.

The checkpoint-mediated control of DNA replication is essential for maintaining the stability of the genome and preventing cancer in humans. The RecQ family of helicases has been shown to be important for the maintenance of genomic integrity in organisms ranging from bacteria to man. We propose that the RecQ homologue, Sgs1p, has an important function in the S-phase checkpoint response of budding yeast, where it may be both a 'sensor' for damage during replication and a 'resolvase' for structures that arise at paused forks. RecQ helicases may serve a unique function that integrates checkpoint proteins with the recombination and replication fork machinery.

Adenosine Triphosphatases↗

PubMed2006

The S. cerevisiae Rrm3p DNA helicase moves with the replication fork and affects replication of all yeast chromosomes.

The Saccharomyces cerevisiae DNA helicase Rrm3p is needed for normal fork progression through >1000 discrete sites scattered throughout the genome. Here we show that replication of all yeast chromosomes was markedly delayed in rrm3 cells. Delayed replication was seen even in a region that lacks any predicted Rrm3p-dependent sites. Based on the pattern of replication intermediates in two-dimensional gels, the rate of fork movement in rrm3 cells appeared similar to wild-type except at known Rrm3p-dependent sites. These data suggest that although Rrm3p has a global role in DNA replication, its activity is needed only or primarily at specific, difficult-to-replicate sites. By the criterion of chromatin immunoprecipitation, Rrm3p was associated with both Rrm3p-dependent and -independent sites, and moved with the replication fork through both. In addition, Rrm3p interacted with Pol2p, the catalytic subunit of DNA polymerase epsilon, in vivo. Thus, rather than being recruited to its sites of action when replication forks stall at these sites, Rrm3p is likely a component of the replication fork apparatus.

Cell Cycle Proteins↗

PubMed2026

R-loops and D-loops: a delicate balance in genomic stability and instability.

R-loops and D-loops are three-stranded nucleic acid structures that have emerged as central regulators of genome stability, gene expression, and DNA metabolism. R-loops form co-transcriptionally or post-transcriptionally when nascent RNA re-anneals with the template DNA strand, generating an RNA: DNA hybrid that displaces the non-template strand into a single-stranded state. These structures are enriched at CpG island promoters, transcription termination sites, and immunoglobulin class-switch regions, where they coordinate transcription regulation, chromatin remodeling, and DNA damage signaling. D-loops are formed when a single-stranded DNA segment pairs with one strand of a duplex and displaces the other, arising through context-dependent mechanisms that include RAD51- or DMC1-mediated strand invasion in homologous recombination, shelterin-assisted invasion at telomeres, and replication-coupled strand displacement at the mitochondrial DNA origin. They serve as indispensable intermediates in double-strand break repair, telomere maintenance, and mitochondrial DNA replication. Recent cryo-electron microscopy studies have resolved the stepwise RAD51-mediated strand exchange mechanism at near-atomic resolution, substantially advancing structural understanding of D-loop biogenesis. Despite their differences in molecular composition, both structures remodel Watson-Crick base pairing and, when dysregulated, are associated with replication fork stalling, transcription-replication conflicts, and aberrant recombination. This review systematically compares the structural features, formation mechanisms, regulatory networks, and biological functions of R-loops and D-loops, with emphasis on their convergent roles in safeguarding genome integrity. We further discuss rapidly evolving detection technologies and emerging therapeutic strategies targeting these structures in cancer and neurodegeneration, identifying key unresolved questions for future investigation.

Genomic Instability↗

PubMed1986

Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae.

The 2 micron circle is a small double stranded DNA plasmid that occurs at about 60 copies per cell in the nuclei of virtually all strains of Saccharomyces cerevisiae. The plasmid has no apparent phenotypic effect on host cells, and is the basis of many useful vectors for the transformation of yeast. Under certain circumstances, the plasmid is apparently able to replicate more than once per cell cycle; this over-replication allows the maintenance of the plasmid at high copy number. The plasmid has two inverted repeat sequences, and encodes a product that catalyses intra-molecular recombination between these two repeats. Models are proposed whereby recombination leads to copy number amplification. In particular, it is proposed that intra-molecular recombination during replication flips the orientation of one replication fork with respect to the other, so that both forks travel in the same direction around a circular monomer template, generating a large multimer from a monomer and a single initiation of replication.

DNA Replication↗

PubMed2003

Identification of MCM4 as a target of the DNA replication block checkpoint system.

Inhibition of the progression of DNA replication prevents further initiation of DNA replication and allows cells to maintain arrested replication forks, but the proteins that are targets of the replication checkpoint system remain to be identified. We report here that human MCM4, a subunit of the putative DNA replicative helicase, is extensively phosphorylated in HeLa cells when they are incubated in the presence of inhibitors of DNA synthesis or are exposed to UV irradiation. The data presented here indicate that the consecutive actions of ATR-CHK1 and CDK2 kinases are involved in this phosphorylation in the presence of hydroxyurea. The phosphorylation sites in MCM4 were identified using specific anti-phosphoantibodies. Based on results that showed that the DNA helicase activity of the MCM4-6-7 complex is negatively regulated by CDK2 phosphorylation, we suggest that the phosphorylation of MCM4 in the checkpoint control inhibits DNA replication, which includes blockage of DNA fork progression, through inactivation of the MCM complex.

Cell Cycle Proteins↗