Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “replication fork maintenance”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources
PubMed2006

Analyzing the dose-dependence of the Saccharomyces cerevisiae global transcriptional response to methyl methanesulfonate and ionizing radiation.

BACKGROUND: One of the most crucial tasks for a cell to ensure its long term survival is preserving the integrity of its genetic heritage via maintenance of DNA structure and sequence. While the DNA damage response in the yeast Saccharomyces cerevisiae, a model eukaryotic organism, has been extensively studied, much remains to be elucidated about how the organism senses and responds to different types and doses of DNA damage. We have measured the global transcriptional response of S. cerevisiae to multiple doses of two representative DNA damaging agents, methyl methanesulfonate (MMS) and gamma radiation. RESULTS: Hierarchical clustering of genes with a statistically significant change in transcription illustrated the differences in the cellular responses to MMS and gamma radiation. Overall, MMS produced a larger transcriptional response than gamma radiation, and many of the genes modulated in response to MMS are involved in protein and translational regulation. Several clusters of coregulated genes whose responses varied with DNA damaging agent dose were identified. Perhaps the most interesting cluster contained four genes exhibiting biphasic induction in response to MMS dose. All of the genes (DUN1, RNR2, RNR4, and HUG1) are involved in the Mec1p kinase pathway known to respond to MMS, presumably due to stalled DNA replication forks. The biphasic responses of these genes suggest that the pathway is induced at lower levels as MMS dose increases. The genes in this cluster with a threefold or greater transcriptional response to gamma radiation all showed an increased induction with increasing gamma radiation dosage. CONCLUSION: Analyzing genome-wide transcriptional changes to multiple doses of external stresses enabled the identification of cellular responses that are modulated by magnitude of the stress, providing insights into how a cell deals with genotoxicity.

DNA Damage↗
PubMed2004

UV-induced ataxia-telangiectasia-mutated and Rad3-related (ATR) activation requires replication stress.

Ataxia-telangiectasia-mutated and Rad3-related (ATR) plays an essential role in the maintenance of genome integrity and cell viability. The kinase is activated in response to DNA damage and initiates a checkpoint signaling cascade by phosphorylating a number of downstream substrates including Chk1. Unlike ataxia-telangiectasia-mutated (ATM), which appears to be mainly activated by DNA double-strand breaks, ATR can be activated by a variety of DNA damaging agents. However, it is still unclear what triggers ATR activation in response to such diverse DNA lesions. One model proposes that ATR can directly recognize DNA lesions, while other recent data suggest that ATR is activated by a common single-stranded DNA (ssDNA) intermediate generated during DNA repair. In this study, we show that UV lesions do not directly activate ATR in vivo. In addition, ssDNA lesions created during the repair of UV damage are also not sufficient to activate the ATR-dependent pathway. ATR activation is only observed in replicating cells indicating that replication stress is required to trigger the ATR-mediated checkpoint cascade in response to UV irradiation. Interestingly, H2AX appears to be required for the accumulation of ATR at stalled replication forks. Together our data suggest that ssDNA at arrested replication forks recruits ATR and initiates ATR-mediated phosphorylation of H2AX and Chk1. Phosphorylated H2AX might further facilitate ATR activation by stabilizing ATR at the sites of arrested replication forks.

Cell Cycle Proteins↗
PubMed2001

UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein.

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Amino Acid Sequence↗
PubMed2005

A second human Dbf4/ASK-related protein, Drf1/ASKL1, is required for efficient progression of S and M phases.

Cdc7-Dbf4 kinase is conserved through evolution and regulates initiation and progression of DNA replication. In human, ASK/hsDbf4 binds and activates huCdc7 during S phase and this kinase complex is essential for DNA replication and cell proliferation. Drf1/ASKL1, a second human Dbf4/ASK-related protein, shares three conserved Dbf4 motifs previously identified on all of the Dbf4-related molecules. Drf1/ASKL1 can bind and activate huCdc7, and Cdc7-ASKL1 complex phosphorylates MCM2. ASKL1 transcription and protein levels oscillate during cell cycle and increase at late S to G2/M phases. The protein is detected predominantly in the nuclear-soluble fraction but not in the chromatin-bound fraction. Inhibition of Drf1/ASKL1 expression by siRNA results in attenuation of cell growth and in the increase of late S and G2/M phase population. siRNA treatment on synchronized cell population revealed that S phase progression is delayed when ASKL1 protein level is decreased. S phase delay may be linked to replication fork block, because increased levels of gammaH2AX and activated form of Chk2 are detected with ASKL1 siRNA in the absence of any additional DNA damages. Furthermore, mitotic progression is retarded in ASKL1 or Cdc7 siRNA-treated cells. Our results suggest that ASKL1 in a complex with Cdc7 may play a role in normal progression of both S and M phases.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Chromatin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication.

A Xenopus homologue of Schizosaccharomyces pombe cdc21 has been characterized as a new member of the MCM family of proteins. The cdc21 protein exhibits cell-cycle dependent chromatin binding and phosphorylation in association with S-phase control. Cdc21 binds to decondensing chromatin at the end of mitosis, localizing to numerous foci which form prior to reconstitution of the nuclear membrane. The association of cdc21 with chromatin occurs in membrane-free high speed extracts and is resistant to detergent extraction. The spatial organization of the cdc21 foci resembles that of pre-replication centres though no co-localization with RP-A was observed. Cdc21 remains bound to chromatin during the initiation of DNA replication and is displaced as the DNA replication forks progress. These subnuclear changes in localization correlate with cell-cycle-regulated changes in phosphorylation. Cdc21 binds to chromatin in an underphosphorylated state, but in early S phase the nuclear localized cdc21 is partially phosphorylated before it is displaced from the chromatin. Cytoplasmic cdc21 remains underphosphorylated but at the beginning of mitosis the entire pool of cdc21 is hyperphosphorylated, possibly by the cdc2/cyclin B kinase. These properties identify Xenopus cdc21 as a possible component of the DNA licensing factor.

Animals↗
PubMed2005

Wild-type p53 stimulates homologous recombination upon sequence-specific binding to the ribosomal gene cluster repeat.

p53 plays a central role in the maintenance of the genome integrity, both as a gatekeeper and a caretaker. Sequence-specific recognition of DNA is underlying the ability of p53 to transcriptionally transactivate target genes during checkpoint control and to regulate DNA replication at the TGCCT repeat from the ribosomal gene cluster (RGC). In contrast, suppression of recombination by p53 has been observed with nonconsensus DNA sequences. In this study, we discovered that wild-type p53 stimulates homologous recombination adjacent to the RGC repeat, whereas downregulation is seen with a mutated version thereof and with a microsatellite repeat sequence. Analysis of the causes possibly underlying the enhancement of homologous recombination revealed that p53 binding to the RGC element delays DNA synthesis. This was demonstrated after integration of the corresponding DNA fragments into our Simian virus 40-based model system, which was used to study recombination on replicating minichromosomes. Differently, with plasmid-based substrates, p53 did not stimulate recombination at the RGC sequence. Thus, in combination with our previous findings, p53 may promote homologous recombination by two separate mechanisms involving either molecular interactions with topoisomerase I or/and by specific binding to certain genomic regions, thereby causing replication fork stalling and recombination.

DNA↗
PubMed2004

Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis.

The structural maintenance of chromosomes (SMC) family of proteins play key roles in the organization, packaging, and repair of chromosomes. Cohesin (Smc1+3) holds replicated sister chromatids together until mitosis, condensin (Smc2+4) acts in chromosome condensation, and Smc5+6 performs currently enigmatic roles in DNA repair and chromatin structure. The SMC heterodimers must associate with non-SMC subunits to perform their functions. Using both biochemical and genetic methods, we have isolated a novel subunit of the Smc5+6 complex, Nse3. Nse3 is an essential nuclear protein that is required for normal mitotic chromosome segregation and cellular resistance to a number of genotoxic agents. Epistasis with Rhp51 (Rad51) suggests that like Smc5+6, Nse3 functions in the homologous recombination based repair of DNA damage. We previously identified two non-SMC subunits of Smc5+6 called Nse1 and Nse2. Analysis of nse1-1, nse2-1, and nse3-1 mutants demonstrates that they are crucial for meiosis. The Nse1 mutant displays meiotic DNA segregation and homologous recombination defects. Spore viability is reduced by nse2-1 and nse3-1, without affecting interhomolog recombination. Finally, genetic interactions shared by the nse mutants suggest that the Smc5+6 complex is important for replication fork stability.

Amino Acid Sequence↗
PubMed2005

The ATM-dependent DNA damage signaling pathway.

Many of the insights that we have gained into the mechanisms involved in cellular DNA damage response pathways have come from studies of human cancer susceptibility syndromes that are altered in DNA damage responses. ATM, the gene mutated in the disorder, ataxia-telangiectasia, is a protein kinase that is a central mediator of responses to DNA double-strand breaks in cells. Recent studies have elucidated the mechanism by which DNA damage activates the ATM kinase and initiates these critical cellular signaling pathways. The SMC1 protein appears to be a particularly important target of the ATM kinase, playing critical roles in controlling DNA replication forks and DNA repair after the damage. A major role for the NBS1 and BRCA1 proteins appears to be in the recruitment of an activated ATM kinase molecule to the sites of DNA breaks so that ATM can phosphorylate SMC1. Generation of mice and cells that are unable to phosphorylate SMC1 demonstrated the importance of SMC1 phosphorylation in the DNA-damage-induced S-phase checkpoint, in determining rates of repair of chromosomal breaks, and in determining cell survival after DNA damage. Focusing on ATM and SMC1, the molecular controls of these pathways is discussed.

Animals↗
PubMed2003

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.

Antigens, Polyomavirus Transforming↗
PubMedDate not supplied

Levels of MCM4 phosphorylation and DNA synthesis in DNA replication block checkpoint control.

Blockage of a DNA replication fork movement not only stabilizes the fork structure but also prevents initiation of DNA replication. We reported that MCM4, a subunit of a putative replicative DNA helicase, is extensively phosphorylated in the presence of hydroxyurea (HU) or after exposure to UV irradiation. Here we examined the relationship between levels of MCM4 phosphorylation and DNA synthesis during DNA replication checkpoint control and after release of the control. The results suggest that there is roughly inverse correlation between these two levels; namely the higher the level of MCM4 phosphorylation, the lower the level of DNA synthesis. The presence of HU or UV irradiation can stimulate phosphorylation at several cyclin-dependent kinase (CDK) sites in MCM4, which can lead to inhibition of MCM4/6/7 helicase activity. These results are consistent with the notion that the phosphorylation of MCM4 is involved in regulation of DNA synthesis in the checkpoint control.

Binding Sites↗
PubMed2002

The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells.

We investigated the organization of DNA replication sites in primary (young or presenescent), immortalized and transformed mammalian cells. Four different methods were used to visualize replication sites: in vivo pulse-labeling with 5-bromo-2'-deoxyuridine (BrdU), followed by either acid depurination, or incubation in nuclease cocktail to expose single-stranded BrdU-substituted DNA regions for immunolabeling; biotin-dUTP labeling of nascent DNA by run-on replication within intact nuclei and staining with fluorescent streptavidin; and, finally, immunolabeling of the replication fork proteins PCNA and RPA. All methods produced identical results, demonstrating no fundamental differences in the spatio-temporal organization of replication patterns between primary, immortal or transformed mammalian cells. In addition, we did not detect a spatial coincidence between the early firing replicons and nuclear lamin proteins, the retinoblastoma protein or the nucleolus in primary human and rodent cells. The retinoblastoma protein does not colocalize in vivo with members of the Mcm family of proteins (Mcm2, 3 and 7) at any point of the cell cycle and neither in the chromatin-bound nor in the soluble nucleoplasmic fraction. These results argue against a direct role for the retinoblastoma or nuclear lamin proteins in mammalian DNA synthesis under normal physiological conditions.

Animals↗
PubMed2001

Initiating DNA synthesis: from recruiting to activating the MCM complex.

The exact duplication of a genome once per cell division is required of every proliferating cell. To achieve this goal, eukaryotes adopt a strategy that limits every replication origin to a single initiation event within a narrow window of the cell cycle by temporally separating the assembly of the pre-replication complex (pre-RC) from the initiation of DNA synthesis. A key component of the pre-RC is the hexameric MCM complex, which is also the presumed helicase of the growing forks. An elaborate mechanism recruits the MCM complex to replication origins, and a regulatory chain reaction converts the poised, but inactive, MCM complex into an enzymatically active helicase. A growing list of proteins, including Mcm10 and Cdt1, are involved in the recruitment process. Two protein kinases, the Cdc7-Dbf4 kinase (DDK) and the cyclin-dependent kinase (CDK), trigger a chain reaction that results in the phosphorylation of the MCM complex and finally in the initiation of DNA synthesis. A composite picture from recent studies suggests that DDK is recruited to the pre-RC during G1 phase but must wait until S phase to phosphorylate the MCM complex. CDK is required for the recruitment of Cdc45 and other downstream components of the elongation machinery.

Animals↗
PubMed2005

The MCM complex: its role in DNA replication and implications for cancer therapy.

The MCM complex controls the once per cell cycle DNA replication in eukaryotic cells. In a process known as DNA replication licensing, it primes chromatin for DNA replication by binding origins of DNA replication during the late M to early G1 phase of the cell cycle. Activated by S phase promoting protein kinases, the origin-bound MCM complexes unwind the double stranded DNA at the origins, recruit DNA polymerases and initiate DNA synthesis. Coupled with the initiation of DNA replication in the S phase, the MCM complexes move away from replication origins as a component of the DNA replication fork, likely serving as DNA helicases. Their departure deprives replication origins the ability to re-initiate DNA replication for the reminder of the cell cycle. Because of its vital role in genome duplication in proliferating cells, deregulation of the MCM function results in chromosomal defects that may contribute to tumorigenesis. The MCM proteins are highly expressed in malignant human cancers cells and pre-cancerous cells undergoing malignant transformation. They are not expressed in differentiated somatic cells that have been withdrawn from the cell cycle. Therefore, these proteins are ideal diagnostic markers for cancer and promising targets for anti-cancer drug development. In this article, I will overview the structures and functions of the MCM complex with an effort to integrate insights from recent biochemical and structural studies. Discussions will also cover activities and structures of the complex that may be useful for the development of drug screens.

Animals↗
PubMed1986

Delayed DNA methylation is an integral feature of DNA replication in mammalian cells.

In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.

Animals↗
PubMed2005

SMC1 involvement in fragile site expression.

Common fragile sites have been involved in neoplastic transformation, although their molecular basis is still poorly understood. Here, we demonstrate that inhibition of the SMC1 by RNAi is sufficient to induce fragile site expression. By investigating normal, ATM- and ATR-deficient cell lines, we provide evidence that the contribution of SMC1 in preventing the collapse of stalled replication fork is an Atr-dependent pathway. Using a fluorescent antibody specific for gamma-H2AX, we show that very rare discrete nuclear foci appear 1 and 2 h after exposure to aphidicolin and/or RNAi-SMC1, but became more numerous and distinct after longer treatment times. In this context, fragile sites might be viewed as an in vitro phenomenon originating from double-strand breaks formed because of a stalled DNA replication that lasted too long to be managed by physiological rescue acting through the Atr/Smc1 axis. We propose that in vivo, following an extreme replication block, rare cells could escape checkpoint mechanisms and enter mitosis with a defect in genome assembly, eventually leading to neoplastic transformation.

Aphidicolin↗
PubMed2000

Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae.

BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2-7 protein complex that may be functioning as the replication-licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks. RESULTS: We show that an mcm10 mutation causes a slow progression of DNA synthesis and a loss of chromosome integrity during the S phase and prevents entry into mitosis, despite apparent completion of chromosomal DNA replication at nonpermissive temperatures. Furthermore, Mcm10p interacts genetically with the origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerases epsilon and delta. Mcm10p is an abundant protein (approximately 4 x 10(4) copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fractions during all phases of the cell cycle. When it is visualized by the chromosome-spreading method followed by immunostaining, Mcm10p forms punctate foci on chromatin throughout the cell cycle and these foci mostly overlap with those of Orc1p, a component of ORC. CONCLUSIONS: These results suggest that Mcm10p, like the Mcm2-7 proteins, is a critical component of the prereplication chromatin and acts together with ORC during the initiation of chromosomal DNA replication; in addition, Mcm10p plays an important role during the elongation of DNA replication.

Cell Cycle Proteins↗
PubMed1998

Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin- and Dbf4-dependent kinases.

Eukaryotic cells use multiple replication origins to replicate their large genomes. Some origins fire early during S phase whereas others fire late. In Saccharomyces cerevisiae, initiator sequences (ARSs) are bound by the origin recognition complex (ORC). Cdc6p synthesized at the end of mitosis joins ORC and facilitates recruitment of Mcm proteins, which renders origins competent to fire. However, origins fire only upon the subsequent activation of S phase cyclin-dependent kinases (S-CDKs) and Dbf4/Cdc7 at the G1/S boundary. We have used a chromatin immunoprecipitation assay to measure the association with ARS sequences of DNA primase and the single-stranded DNA binding replication protein A (RPA) when fork movement is inhibited by hydroxyurea (HU). RPA's association with origins requires S-CDKs, Dbf4/Cdc7 kinase and an Mcm protein. The recruitment of DNA primase depends on RPA. Furthermore, early- and late-firing origins differ not in the timing of their recruitment of an Mcm protein, but in the timing of RPA's recruitment. RPA is recruited to early but not to late origins in HU. We also show that Rad53 kinase is required to prevent RPA association with a late origin in HU. Our data suggest that the origin unwinding accompanied by RPA association is a key step, regulated by S-CDKs, Dbf4/Cdc7 and Rad53p. Thus, in the presence of active S-CDKs and Dbf4/Cdc7, Mcms may open origins and thereby facilitate the loading of RPA.

Adenosine Triphosphatases↗
PubMed2002

Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex.

Mcm, which is composed of six structurally related subunits (Mcm2-7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the Mcm4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the Mcm4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the Mcm4/6/7 complex containing a zinc finger mutant of Mcm4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The Mcm4/6/7 complex containing this Mcm4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of Mcm4 is involved in subunit interactions of trimers. The Mcm4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric Mcm4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003-8015), our data indicate distinct roles of Mcm4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the Mcm4/6/7 complex.

Adenosine Triphosphatases↗