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Results for “replication fork maintenance”

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PubMed2001

Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures.

The minichromosome maintenance (Mcm) proteins 2-7 are required for both the initiation and elongation steps of chromosomal DNA replication. Previous studies have shown that the Mcm complex consisting of the Mcm 4, 6, and 7 proteins contains 3' to 5' DNA helicase activity with limited processivity (displacing duplex DNA regions up to 30 nt). In this report, we show that the presence of both 5' and 3' single-stranded tails in DNA helicase substrates is essential for the processive helicase activity of the Mcm complex. The presence of both 5' and 3' tails facilitated the formation of double heterohexameric complexes of Mcm4/6/7 on substrate DNA, which appeared to be essential for the processive helicase activity. The double heterohexameric complex of Mcm4/6/7, in the presence of a single-strand DNA binding protein, is capable of unwinding duplex DNA region of about 600 bp in length. These results support the hypothesis that the Mcm4/6/7 complex can function as a replication helicase.

Cell Cycle Proteins↗
PubMed1998

Dna2 of Saccharomyces cerevisiae possesses a single-stranded DNA-specific endonuclease activity that is able to act on double-stranded DNA in the presence of ATP.

To gain further insights into the biological functions of Dna2, previously known as a cellular replicative helicase in Saccharomyces cerevisiae, we examined biochemical properties of the recombinant Dna2 protein purified to homogeneity. Besides the single-stranded (ss) DNA-dependent ATPase activity as reported previously, we were able to demonstrate that ssDNA-specific endonuclease activity is intrinsically associated with Dna2. Moreover, Dna2 was capable of degrading duplex DNA in an ATP-dependent fashion. ATP and dATP, the only nucleotides hydrolyzed by Dna2, served to stimulate Dna2 to utilize duplex DNA, indicating their hydrolysis is required. Dna2 was able to unwind short duplex only under the condition where the endonuclease activity was minimized. This finding implies that Dna2 unwinds only partially the 3'-end of duplex DNA and generates a stretch of ssDNA of limited length, which is subsequently cleaved by the ssDNA-specific endonuclease activity. A point mutation at the conserved ATP-binding site of Dna2 inactivated concurrently ssDNA-dependent ATPase, ATP-dependent nuclease, and helicase activities, indicating that they all reside in Dna2 itself. By virtue of its nucleolytic activities, the Dna2 protein may function in the maintenance of chromosomal integrity, such as repair or other related process, rather than in propagation of cellular replication forks.

Adenosine Triphosphate↗
PubMed2003

Evidence for a role of MCM (mini-chromosome maintenance)5 in transcriptional repression of sub-telomeric and Ty-proximal genes in Saccharomyces cerevisiae.

The MCM (mini-chromosome maintenance) genes have a well established role in the initiation of DNA replication and in the elongation of replication forks in Saccharomyces cerevisiae. In this study we demonstrate elevated expression of sub-telomeric and Ty retrotransposon-proximal genes in two mcm5 strains. This pattern of up-regulated genes resembles the genome-wide association of MCM proteins to chromatin that was reported earlier. We link the altered gene expression in mcm5 strains to a reversal of telomere position effect (TPE) and to remodeling of sub-telomeric and Ty chromatin. We also show a suppression of the Ts phenotype of a mcm5 strain by the high copy expression of the TRA1 component of the chromatin-remodeling SAGA/ADA (SPT-ADA-GCN5 acetylase/ADAptor). We propose that MCM proteins mediate the establishment of silent chromatin domains around telomeres and Ty retrotransposons.

Blotting, Northern↗
PubMed2006

Characterization of the helicase activity and substrate specificity of Mycobacterium tuberculosis UvrD.

UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologue was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. Oligonucleotides as short as four nucleotides were sufficient to promote the ATPase activity. The DNA helicase activity of the enzyme was only fueled by ATP and dATP. UvrD preferentially unwound 3'-single-stranded tailed duplex substrates over 5'-single-stranded ones, indicating that the protein had a duplex-unwinding activity with 3'-to-5' polarity. A 3' single-stranded DNA tail of 18 nucleotides was required for effective unwinding. By using a series of synthetic oligonucleotide substrates, we demonstrated that M. tuberculosis UvrD has an unwinding preference towards nicked DNA duplexes and stalled replication forks, representing the likely sites of action in vivo. The potential role of M. tuberculosis UvrD in maintenance of bacterial genomic integrity makes it a promising target for drug design against M. tuberculosis.

Adenosine Triphosphatases↗
PubMed2003

The DNA polymerase alpha-primase complex: multiple functions and interactions.

DNA polymerase alpha (pol alpha) holds a special position among the growing family of eukaryotic DNA polymerases. In fact, pol alpha is associated with DNA primase to form a four subunit complex and, as a consequence, is the only enzyme able to start DNA synthesis de novo. Because of this peculiarity the major role of the DNA polymerase alpha-primase complex (pol-prim) is in the initiation of DNA replication at chromosomal origins and in the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. However, pol-prim seems to play additional roles in other complex cellular processes, such as the response to DNA damage, telomere maintenance, and the epigenetic control of higher order chromatin assembly.

Animals↗
PubMed2026

Distinct functions of mammalian RAD51 paralogs in genome maintenance.

RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are evolutionarily conserved essential proteins for cell survival and genome maintenance. RAD51 paralogs were originally identified to play a role in homologous recombination-mediated repair of DNA double-strand breaks (DSBs). However, investigations over the last decade have uncovered new roles of RAD51 paralogs beyond DSB repair in replication stress responses, including replication fork progression, fork stability, and its restart. Recent structural studies have not only uncovered the molecular architecture of previously known RAD51 paralog complexes but also identified novel paralog complex assemblies, providing mechanistic insights into their various genome-maintenance functions. Additionally, a role for RAD51 paralogs in resolving R-loops has been identified, and studies with cancer-associated variants suggest that RAD51 paralogs are potential determinants of cancer susceptibility and therapeutic responses. In the present review, we highlight the recently deciphered structures and novel functions of RAD51 paralog complexes and discuss the clinical and therapeutic implications.

Rad51 Recombinase↗
PubMed2005

Multifaceted role of the Saccharomyces cerevisiae Srs2 helicase in homologous recombination regulation.

Homologous recombination (HR) is a major pathway for the elimination of DNA DSBs (double-strand breaks) induced by high-energy radiation and chemicals, or that arise due to endogenous damage and stalled DNA replication forks. If not processed properly, DSBs can lead to cell death, chromosome aberrations and tumorigenesis. Even though HR is important for genome maintenance, it can also interfere with other DNA repair mechanisms and cause gross chromosome rearrangements. In addition, HR can generate DNA or nucleoprotein intermediates that elicit prolonged cell-cycle arrest and sometimes cell death. Genetic analyses in the yeast Saccharomyces cerevisiae have revealed a central role of the Srs2 helicase in preventing untimely HR events and in inhibiting the formation of potentially deleterious DNA structures or nucleoprotein complexes upon DNA replication stress. Paradoxically, efficient repair of DNA DSBs by HR is dependent on Srs2. In this paper, we review recent molecular studies aimed at deciphering the multifaceted role of Srs2 in HR and other cellular processes. These studies have provided critical insights into how HR is regulated in order to preserve genomic integrity and promote cell survival.

Cytoskeleton↗
PubMed2004

The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites.

In the cell, Dnmt1 is the major enzyme in maintenance of the pattern of DNA methylation after DNA replication. Evidence suggests that the protein is located at the replication fork, where it could directly modify nascent DNA immediately after replication. To elucidate the potential mechanism of this process, we investigate the processivity of DNA methylation and accuracy of copying an existing pattern of methylation in this study using purified Dnmt1 and hemimethylated substrate DNA. We demonstrate that Dnmt1 methylates a hemimethylated 958-mer substrate in a highly processive reaction. Fully methylated and unmethylated CG sites do not inhibit processive methylation of the DNA. Extending previous work, we show that unmethylated sites embedded in a hemimethylated context are modified at an approximately 24-fold reduced rate, which demonstrates that the enzyme accurately copies existing patterns of methylation. Completely unmodified DNA is methylated even more slowly due to an allosteric activation of Dnmt1 by methylcytosine-containing DNA. Interestingly, Dnmt1 is not able to methylate hemimethylated CG sites on different strands of the DNA in a processive manner, indicating that Dnmt1 keeps its orientation with respect to the DNA while methylating the CG sites on one strand of the DNA.

Animals↗
PubMed2006

The archaeal Hjm helicase has recQ-like functions, and may be involved in repair of stalled replication fork.

The archaeal Hjm is a structure-specific DNA helicase, which was originally identified in the hyperthermophilic archaeon, Pyrococcus furiosus, by in vitro screening for Holliday junction migration activity. Further biochemical analyses of the Hjm protein from P. furiosus showed that this protein preferably binds to fork-related Y-structured DNAs and unwinds their double-stranded regions in vitro, just like the E. coli RecQ protein. Furthermore, genetic analyses showed that Hjm produced in E. coli cells partially complemented the defect of functions of RecQ in a recQ mutant E. coli strain. These results suggest that Hjm may be a functional counterpart of RecQ in Archaea, in which it is necessary for the maintenance of genome integrity, although the amino acid sequences are not conserved. The functional interaction of Hjm with PCNA for its helicase activity further suggests that the Hjm works at stalled replication forks, as a member of the reconstituted replisomes to restart replication.

Amino Acid Sequence↗
PubMed2004

Werner syndrome protein and the MRE11 complex are involved in a common pathway of replication fork recovery.

Werner syndrome (WS) is an autosomal recessive disease that predisposes individuals to a wide range of cancers. The gene mutated in WS, WRN, encodes a member of the RecQ family of DNA helicases. The precise DNA metabolic processes in which WRN participates remain to be elucidated. However, it has been proposed that WRN might play an important role in the maintenance of genetic stability during DNA replication, possibly cooperating with other proteins. Here, we show that, following DNA replication arrest, WRN associates and colocalizes with the MRE11 complex at PCNA sites. We also provide evidence that both WRN/MRE11 complex association and proper WRN relocalization after HU treatment require a functional MRE11 complex. We demonstrate that mutations altering the functionality of WRN or that of the MRE11 complex result in chromosomal breakage during DNA replication and enhanced cell death following replication arrest. Finally, we show that the DNA breakage in replicating cells and apoptosis observed in WS are not enhanced by concomitant knock down of MRE11 by RNAi, indicating that WRN and MRE11 complex act in a common pathway. These results suggest a functional relationship between WRN and the MRE11 complex in response to replication fork arrest, disclosing a common action of WRN and the MRE11 complex in the pathway(s) preserving genome stability during DNA replication.

Apoptosis↗
PubMed2003

Histone deposition at the replication fork: a matter of urgency.

In this issue of Molecular Cell, Ye et al. provide a biological rationale for rapid histone deposition behind the replication fork. They show that defects in nucleosome assembly lead to DNA double-strand breaks and S phase arrest. Their results have important implications for the maintenance of genome integrity in proliferating cells.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2003

Reconstitution of recombination-dependent DNA synthesis in herpes simplex virus 1.

The repair of double-strand DNA breaks by homologous recombination is essential for the maintenance of genome stability. In herpes simplex virus 1, double-strand DNA breaks may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection. Double-strand DNA breaks are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization. We have reconstituted a system using purified proteins in which strand invasion is coupled with DNA synthesis. In this system, the viral single-strand DNA-binding protein promotes assimilation of single-stranded DNA into a homologous supercoiled plasmid, resulting in the formation of a displacement loop. The 3' terminus of the invading DNA serves as a primer for long-chain DNA synthesis promoted by the viral DNA replication proteins, including the polymerase and helicase-primase. Efficient extension of the invading primer also requires a DNA-relaxing enzyme (eukaryotic topoisomerase I or DNA gyrase). The viral polymerase by itself is insufficient for DNA synthesis, and a DNA-relaxing enzyme cannot substitute for the viral helicase-primase. The viral single-strand DNA-binding protein, in addition to its role in the invasion process, is also required for long-chain DNA synthesis. Form X, a topologically distinct, positively supercoiled form of displacement-loop, does not serve as a template for DNA synthesis. These observations support a model in which recombination and replication contribute toward maintaining viral genomic stability by repairing double-strand breaks. They also account for the extensive branching observed during viral replication in vivo.

DNA Replication↗
PubMed2006

Interaction between PCNA and diubiquitinated Mcm10 is essential for cell growth in budding yeast.

The minichromosome maintenance protein 10 (Mcm10) is an evolutionarily conserved factor that is essential for replication initiation and elongation. Mcm10 is part of the eukaryotic replication fork and interacts with a variety of proteins, including the Mcm2-7 helicase and DNA polymerase alpha/primase complexes. A motif search revealed a match to the proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) box in Mcm10. Here, we demonstrate a direct interaction between Mcm10 and PCNA that is alleviated by mutations in conserved residues of the PIP box. Interestingly, only the diubiquitinated form of Mcm10 binds to PCNA. Diubiquitination of Mcm10 is cell cycle regulated; it first appears in late G(1) and persists throughout S phase. During this time, diubiquitinated Mcm10 is associated with chromatin, suggesting a direct role in DNA replication. Surprisingly, a Y245A substitution in the PIP box of Mcm10 that inhibits the interaction with PCNA abolishes cell proliferation. This severe-growth phenotype, which has not been observed for analogous mutations in other PCNA-interacting proteins, is rescued by a compensatory mutation in PCNA that restores interaction with Mcm10-Y245A. Taken together, our results suggest that diubiquitinated Mcm10 interacts with PCNA to facilitate an essential step in DNA elongation.

Cell Cycle↗
PubMed2003

Werner protein stimulates topoisomerase I DNA relaxation activity.

Werner syndrome (WS) is a human premature aging disorder characterized by the early onset of age-related clinical features and an elevated incidence of cancer. The Werner protein (WRN) belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. Potential cooperation between RecQ helicases and topoisomerases in many aspects of DNA metabolism, such as the progression of replication forks, transcription, recombination, and repair, has been reported. Here, we show a physical and functional interaction between WRN and topoisomerase I (topo I). WRN colocalizes and interacts directly with topo I. WRN stimulates the ability of topo I to relax negatively supercoiled DNA and specifically stimulates the religation step of the relaxation reaction. Moreover, cell extracts from WS fibroblasts exhibit a decrease in the relaxation activity of negatively supercoiled DNA. We have identified two regions of WRN that mediate functional interaction with topo I, and they are located at the NH(2) and COOH termini of the WRN protein. In a reciprocal functional interaction, topo I inhibits the ATPase activity of WRN. Our data provide new insight into the interrelationship between RecQ helicases and topoisomerases in the maintenance of genomic integrity and prevention of tumorigenesis.

Adenosine Triphosphatases↗
PubMed2005

Preventing re-replication of chromosomal DNA.

To ensure its duplication, chromosomal DNA must be precisely duplicated in each cell cycle, with no sections left unreplicated, and no sections replicated more than once. Eukaryotic cells achieve this by dividing replication into two non-overlapping phases. During late mitosis and G1, replication origins are 'licensed' for replication by loading the minichromosome maintenance (Mcm) 2-7 proteins to form a pre-replicative complex. Mcm2-7 proteins are then essential for initiating and elongating replication forks during S phase. Recent data have provided biochemical and structural insight into the process of replication licensing and the mechanisms that regulate it during the cell cycle.

Animals↗
PubMed2023

H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins.

The transmission and maintenance of genetic information in eukaryotic cells relies on the faithful duplication of the entire genome. In each round of division, excessive replication origins are licensed, with only a fraction activated to give rise to bi-directional replication forks in the context of chromatin. However, it remains elusive how eukaryotic replication origins are selectively activated. Here we demonstrate that O-GlcNAc transferase (OGT) enhances replication initiation by catalyzing H4S47 O-GlcNAcylation. Mutation of H4S47 impairs DBF4-dependent protein kinase (DDK) recruitment on chromatin, causing reduced phosphorylation of the replicative helicase mini-chromosome maintenance (MCM) complex and compromised DNA unwinding. Our short nascent-strand sequencing results further confirm the importance of H4S47 O-GlcNAcylation in origin activation. We propose that H4S47 O-GlcNAcylation directs origin activation through facilitating MCM phosphorylation, and this may shed light on the control of replication efficiency by chromatin environment.

Animals↗
PubMed2006

Condensin loaded onto the replication fork barrier site in the rRNA gene repeats during S phase in a FOB1-dependent fashion to prevent contraction of a long repetitive array in Saccharomyces cerevisiae.

An average of 200 copies of the rRNA gene (rDNA) is clustered in a long tandem array in Saccharomyces cerevisiae. FOB1 is known to be required for expansion/contraction of the repeats by stimulating recombination, thereby contributing to the maintenance of the average copy number. In Deltafob1 cells, the repeats are still maintained without any fluctuation in the copy number, suggesting that another, unknown system acts to prevent repeat contraction. Here, we show that condensin acts together with FOB1 in a functionally complemented fashion to maintain the long tandem repeats. Six condensin mutants possessing severely contracted rDNA repeats were isolated in Deltafob1 cells but not in FOB1+ cells. We also found that the condensin complex associated with the nontranscribed spacer region of rDNA with a major peak coincided with the replication fork barrier (RFB) site in a FOB1-dependent fashion. Surprisingly, condensin association with the RFB site was established during S phase and was maintained until anaphase. These results indicate that FOB1 plays a novel role in preventing repeat contraction by regulating condensin association and suggest a link between replication termination and chromosome condensation and segregation.

Adenosine Triphosphatases↗
PubMed2004

Werner syndrome protein, the MRE11 complex and ATR: menage-à-trois in guarding genome stability during DNA replication?

The correct execution of the DNA replication process is crucially import for the maintenance of genome integrity of the cell. Several types of sources, both endogenous and exogenous, can give rise to DNA damage leading to the DNA replication fork arrest. The processes by which replication blockage is sensed by checkpoint sensors and how the pathway leading to resolution of stalled forks is activated are still not completely understood. However, recent emerging evidence suggests that one candidate for a sensor of replication stress is ATR and that, together with a member of RecQ family helicases, Werner syndrome protein (WRN) and MRE11 complex, can collaborate to promote the restarting of DNA synthesis through the resolution of stalled replication forks. Here, we discuss how WRN, the MRE11 complex and the ATR kinase could work together in response to replication blockage to avoid DNA replication fork collapse and genome instability.

DNA Damage↗