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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↗
PubMed1988

Location of sites that inhibit progression of replication forks in the terminus region of Escherichia coli.

We used a Southern hybridization assay to locate precisely the sites at which DNA replication is arrested in the terminus region of the Escherichia coli chromosome. The assay was based on the properties of restriction fragments that contain stalled replication forks. Replication forks that entered the terminus from the clockwise direction with respect to the genetic map were inhibited near manA at a site called T2, which we located at kilobase 442 on the physical map of Bouché (J. P. Bouché, J. Mol. Biol. 154:1-20, 1982). Those that entered the terminus region traveling in the counterclockwise direction were inhibited near pyrF at a site called T1, which we located at kilobase 90. In each case we found only a single, precise site of arrest. Inhibition at T1 was not detectable in our assay in strains lacking the trans-acting locus tus, which is located near T2 and is required for T1 to function. We demonstrated that the sites of inhibition are also used during termination of replication in exponentially growing, wild-type cells. In all previous studies on the terminus of E. coli, inhibition has only been detected in strains that were modified so that the origin used was placed near the terminus to force the use of the sites of inhibition.

Chromosome Mapping↗
PubMed2000

Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress.

The BRCA1 gene encodes a tumor suppressor that is mutated in 50% of familial breast cancers. The BRCA1 protein has been implicated in the DNA damage response, as DNA damage induces the phosphorylation of BRCA1 and causes its recruitment into nuclear foci that contain DNA repair proteins. The ataxia-telangiectasia-mutated (ATM) gene product controls overall BRCA1 phosphorylation in response to gamma-irradiation (IR). In this study, we show that BRCA1 phosphorylation is only partially ATM dependent in response to IR and ATM independent in response to treatment with UV light, or the DNA replication inhibitors hydroxyurea (HU) and aphidicolin (APH). We provide evidence that the kinase responsible for this phosphorylation is the ATM-related kinase, ATR. ATR phosphorylates BRCA1 on six Ser/Thr residues, including Ser 1423, in vitro. Increased expression of ATR enhanced the phosphorylation of BRCA1 on Ser 1423 following cellular exposure to HU or UV light, whereas doxycycline-induced expression of a kinase-inactive ATR mutant protein inhibited HU- or UV light-induced Ser 1423 phosphorylation in GM847 fibroblasts, and partially suppressed the phosphorylation of this site in response to IR. Thus, ATR, like ATM, controls BRCA1 phosphorylation in vivo. Although ATR isolated from DNA-damaged cells does not show enhanced kinase activity in vitro, we found that ATR responds to DNA damage and replication blocks by forming distinct nuclear foci at the sites of stalled replication forks. Furthermore, ATR nuclear foci overlap with the nuclear foci formed by BRCA1. The dramatic relocalization of ATR in response to DNA damage points to a possible mechanism for its ability to enhance the phosphorylation of substrates in response to DNA damage. Together, these results demonstrate that ATR and BRCA1 are components of the same genotoxic stress-responsive pathway, and that ATR directly phosphorylates BRCA1 in response to damaged DNA or stalled DNA replication.

Animals↗
PubMed2001

Hypersensitivity to camptothecin in MSH2 deficient cells is correlated with a role for MSH2 protein in recombinational repair.

DNA mismatch repair (MMR) corrects DNA polymerase insertion errors that have escaped proofreading in order to avoid the accumulation of deleterious mutations. While the role of MMR in the correction of replication errors is well established, its involvement in the processing of DNA damage induced by chemical and physical agents is less clear. A role for some of the MMR proteins, such as MSH2, in the repair of double strand break (DSBs) through recombination has also been envisaged. Why MMR- deficient cells are sensitive to agents causing replication fork stalling and thus DSBs remains unclear. To verify a possible role of MSH2 in homologous recombinational repair, we have treated cells from knockout mice for the MSH2 gene and mouse colorectal carcinoma cells also defective for MSH2 with different doses of camptothecin, an agent known to interfere with DNA replication. In the absence of MSH2, we found a reduced survival rate accompanied by higher levels of chromosomal damage and SCE induction. Furthermore, MSH2(-/-) cells displayed an elevated spontaneous RAD51 focus-forming activity and a higher induction of RAD51 foci following camptothecin treatment. Thus, the absence of MSH2 could result in both spontaneous DNA damage and uncontrolled recombination events leading to the observed higher yield of chromosomal damage and the higher induction of RAD51 foci following CPT treatment. Therefore, our results suggest an involvement of MSH2 in the early events leading to correct RAD51 relocalization after the formation of DSBs specifically produced at the blocked replication fork.

Animals↗
PubMed2006

Rad18 regulates DNA polymerase kappa and is required for recovery from S-phase checkpoint-mediated arrest.

We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polkappa to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polkappa in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polkappa. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polkappa. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polkappa in a DNA damage-independent manner. Therefore, association of Polkappa with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18(-/-) transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18(-/-) cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polkappa. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polkappa to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2001

Topological challenges to DNA replication: conformations at the fork.

The unwinding of the parental DNA duplex during replication causes a positive linking number difference, or superhelical strain, to build up around the elongating replication fork. The branching at the fork and this strain bring about different conformations from that of (-) supercoiled DNA that is not being replicated. The replicating DNA can form (+) precatenanes, in which the daughter DNAs are intertwined, and (+) supercoils. Topoisomerases have the essential role of relieving the superhelical strain by removing these structures. Stalled replication forks of molecules with a (+) superhelical strain have the additional option of regressing, forming a four-way junction at the replication fork. This four-way junction can be acted on by recombination enzymes to restart replication. Replication and chromosome folding are made easier by topological domain barriers, which sequester the substrates for topoisomerases into defined and concentrated regions. Domain barriers also allow replicated DNA to be (-) supercoiled. We discuss the importance of replicating DNA conformations and the roles of topoisomerases, focusing on recent work from our laboratory.

DNA Replication↗
PubMed2006

Indirect mechanisms of genomic instability and the biological significance of mutations at tandem repeat loci.

Radiation induction of genomic instability has two features: induction of untargeted mutation and delayed mutation. These phenomena have been studied mostly in tissue culture cells, but analyses have also been conducted in whole body systems. The study of response in whole body systems frequently applies repeat sequences as markers to detect mutations. These studies have generated conflicting findings. In addition, lack of knowledge of the mechanisms involved in repeat mutation confounds the interpretation of the biological significance of increased rates of repeat mutation. In this review, some of the existing controversies of genomic instability are discussed in relation to the mechanism of repeat mutation. Analyses of published and unpublished studies indicate a mechanistic similarity between radiation-induced genomic instability at repeat loci and dynamic mutations of triplet repeats. Because of their repetitive nature, repeat sequences frequently block progression of replication forks and are consequently resolved by slippage and/or recombination. Irradiation of cells induces S checkpoints and promotes slippage/recombination mediated repeat mutations. Thus, genomic instability at repeat loci might be viewed as a consequence of cellular attempts to restore the stability of replication in the face of the stalled replication fork; this process can occur both spontaneously as well as after exposure to radiation.

Animals↗
PubMed2005

Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations.

The yeast checkpoint kinases Mec1 and Rad53 are required for genomic stability in the presence of replicative stress. When replication forks stall, the stable maintenance of replisome components requires the ATR kinase Mec1/Ddc2 and the RecQ helicase Sgs1. It was unclear whether either Mec1 or Sgs1 action requires the checkpoint effector kinase, Rad53. By combining sgs1Delta with checkpoint-deficient alleles, we can now distinguish the role of Mec1 at stalled forks from that of Rad53. We show that the S-phase-specific mec1-100 allele, like the sgs1Delta mutation, partially destabilizes DNA polymerases at stalled forks, yet combining the mec1-100 and sgs1Delta mutations leads to complete disassociation of the replisome, loss of RPA, irreversible termination of nucleotide incorporation, and compromised recovery from hydroxyurea (HU) arrest. These events coincide with a dramatic increase in both spontaneous and HU-induced chromosomal rearrangements. Importantly, in sgs1Delta cells, RPA levels at stalled forks do not change, although Ddc2 recruitment is compromised, explaining the partial Sgs1 and Mec1 interdependence. Loss of Rad53 kinase, on the other hand, does not affect the levels of DNA polymerases at arrested forks, but leads to MCM protein dissociation. Finally, confirming its unique role during replicative stress, Mec1, and not Tel1, is shown to modify fork-associated histone H2A.

Adenosine Triphosphatases↗
PubMed2002

Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4.

The blockage of replication forks can result in the disassembly of the replicative apparatus and reversal of the fork to form a DNA junction that must be processed in order for replication to restart and sister chromatids to segregate at mitosis. Fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4 are endonucleases that have been implicated in the processing of aberrant DNA junctions formed at stalled replication forks. Here we have investigated the activity of purified Mus81-Eme1 and Mus81-Mms4 on substrates that resemble DNA junctions that are expected to form when a replication fork reverses. Both enzymes cleave Holliday junctions and substrates that resemble normal replication forks poorly or not at all. However, forks where the equivalents of either both the leading and lagging strands or just the lagging strand are juxtaposed at the junction point, or where either the leading or lagging strand has been unwound to produce a fork with a single-stranded tail, are cleaved well. Cleavage sites map predominantly between 3 and 6 bp 5' of the junction point. For most substrates the leading strand template is cleaved. The sole exception is a fork with a 5' single-stranded tail, which is cleaved in the lagging strand template.

DNA↗
PubMed2002

The nonmutagenic repair of broken replication forks via recombination.

When replication forks stall or collapse at sites of DNA damage, there are two avenues for fork rescue. Mutagenic translesion synthesis by a special class of DNA polymerases can move a fork past the damage, but can leave behind mutations. The alternative nonmutagenic pathways for fork repair involve cellular recombination systems. In bacteria, nonmutagenic repair of replication forks may occur as often as once per cell per generation, and is the favored path for fork restoration under normal growth conditions. Replication fork repair is almost certainly the major function of bacterial recombination systems, and was probably the impetus for the evolution of recombination systems. Increasingly, the nonmutagenic repair of replication forks is seen as a major function of eukaryotic recombination systems as well.

Bacteria↗
PubMed2006

Unwinding of forked DNA structures by UvrD.

Many studies have demonstrated the need for processing of blocked replication forks to underpin genome duplication. UvrD helicase in Escherichia coli has been implicated in the processing of damaged replication forks, or the recombination intermediates formed from damaged forks. Here we show that UvrD can unwind forked DNA structures, in part due to the ability of UvrD to initiate unwinding from discontinuities within the phosphodiester backbone of DNA. UvrD does therefore have the capacity to target DNA intermediates of replication and recombination. Such an activity resulted in unwinding of what would be the parental duplex DNA ahead of either a stalled replication fork or a D-loop formed by recombination. However, UvrD had a substrate preference for fork structures having a nascent lagging strand at the branch point but no leading strand. Furthermore, at such structures the polarity of UvrD altered so that unwinding of the lagging strand predominated. This reaction is reminiscent of the PriC-Rep pathway of replication restart, suggesting that UvrD and Rep may have at least partially redundant functions.

Adenosine Triphosphatases↗
PubMed2006

Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos.

In most cells, the DNA damage checkpoint delays cell division when replication is stalled by DNA damage. In early Caenorhabditis elegans embryos, however, the checkpoint responds to developmental signals that control the timing of cell division, and checkpoint activation by nondevelopmental inputs disrupts cell cycle timing and causes embryonic lethality. Given this sensitivity to inappropriate checkpoint activation, we were interested in how embryos respond to DNA damage. We demonstrate that the checkpoint response to DNA damage is actively silenced in embryos but not in the germ line. Silencing requires rad-2, gei-17, and the polh-1 translesion DNA polymerase, which suppress replication fork stalling and thereby eliminate the checkpoint-activating signal. These results explain how checkpoint activation is restricted to developmental signals during embryogenesis and insulated from DNA damage. They also show that checkpoint activation is not an obligatory response to DNA damage and that pathways exist to bypass the checkpoint when survival depends on uninterrupted progression through the cell cycle.

Animals↗
PubMed2006

Homologous recombination is required for genome stability in the absence of DOG-1 in Caenorhabditis elegans.

In C. elegans, DOG-1 prevents deletions that initiate in polyG/polyC tracts (G/C tracts), most likely by unwinding secondary structures that can form in G/C tracts during lagging-strand DNA synthesis. We have used the dog-1 mutant to assay the in vivo contribution of various repair genes to the maintenance of G/C tracts. Here we show that DOG-1 and the BLM ortholog, HIM-6, act synergistically during replication; simultaneous loss of function of both genes results in replicative stress and an increase in the formation of small deletions that initiate in G/C tracts. Similarly, we demonstrate that the C. elegans orthologs of the homologous recombination repair genes BARD1, RAD51, and XPF and the trans-lesion synthesis polymerases poleta and polkappa contribute to the prevention of deletions in dog-1 mutants. Finally, we provide evidence that the small deletions generated in the dog-1 background are not formed through homologous recombination, nucleotide excision repair, or nonhomologous end-joining mechanisms, but appear to result from a mutagenic repair mechanism acting at G/C tracts. Our data support the hypothesis that absence of DOG-1 leads to replication fork stalling that can be repaired by deletion-free or deletion-prone mechanisms.

Animals↗
PubMed2004

Requirement of Rrm3 helicase for repair of spontaneous DNA lesions in cells lacking Srs2 or Sgs1 helicase.

The Rrm3 DNA helicase of Saccharomyces cerevisiae interacts with proliferating cell nuclear antigen and is required for replication fork progression through ribosomal DNA repeats and subtelomeric and telomeric DNA. Here, we show that rrm3 srs2 and rrm3 sgs1 mutants, in which two different DNA helicases have been inactivated, exhibit a severe growth defect and undergo frequent cell death. Cells lacking Rrm3 and Srs2 arrest in the G(2)/M phase of the cell cycle with 2N DNA content and frequently contain only a single nucleus. The phenotypes of rrm3 srs2 and rrm3 sgs1 mutants were suppressed by disrupting early steps of homologous recombination. These observations identify Rrm3 as a new member of a network of pathways, involving Sgs1 and Srs2 helicases and Mus81 endonuclease, suggested to act during repair of stalled replication forks.

Amino Acid Sequence↗
PubMedDate not supplied

Multiple roles of replication forks in S phase checkpoints: sensors, effectors and targets.

There is mounting evidence that replication defects are the major source of spontaneous genomic instability in the cell and that S phase checkpoints are the principle defense against such instability. In Saccharomyces cerevisiae, S phase checkpoints can be provoked by either depletion of dNTPs or DNA damage. In both cases the checkpoint kinases Mec1 and Rad53 act to suppress late origin firing, stabilize slowed or stalled replication forks and prevent S phase progression until conditions are appropriate for the resumption of DNA replication. The present review highlights recent work emphasizing the central importance of replication forks, not just as targets, but also as sensors and primary effectors of checkpoint responses, and identifies the roles played by specific fork-associated factors in these processes.

Animals↗
PubMedDate not supplied

A model for the phenotypic presentation of Werner's syndrome.

Werner's syndrome (WS) is a valuable model of accelerated ageing and results from mutations in a recQ helicase (wrn). WS fibroblasts show a mutator phenotype, replication fork stalling, increased rates of mean telomeric loss and accelerated cellular senescence. Senescence has been proposed as a candidate mechanism for the ageing of mitotic tissue. However, some mitotic tissues (such as the immune system) seem unaffected in WS. Is this evidence against a role for cell senescence in ageing? Two experiments resolve this paradox (i) the demonstration that the abbreviated replicative lifespan of WS fibroblasts can be corrected by the ectopic expression of telomerase and (ii) the demonstration that T cells derived from WS patients have the mutator phenotype characteristic of the disease but show no reduction in replicative potential. Since T cells can upregulate telomerase naturally these findings are consistent with a model in which the only wrn-mediated deletions that have a significant effect on replicative lifespan are those at or near the telomere. These data are thus supportive of a role for senescence in the ageing of the immune system. Emerging data on divisional counting mechanisms have the potential to produce many other apparent WS "paradoxes". Accordingly, we propose a general model for the phenotypic presentation of WS, which includes a modification of the Olovnikov model of telomere erosion. Somewhat unexpectedly, this predicts that accelerated senescence should not be observed in all telomerase-negative WS cell types.

Aging↗
PubMed2003

Escherichia coli cells with increased levels of DnaA and deficient in recombinational repair have decreased viability.

The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, beta clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, beta clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the beta clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 micro m) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional approximately 100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.

Bacterial Proteins↗
PubMed2003

PriA supports two distinct pathways for replication restart in UV-irradiated Escherichia coli cells.

The Escherichia coli PriA protein loads the DnaB replicative helicase at branched DNA structures independently of the replication initiator protein, DnaA, and thereby facilitates assembly of functional replisomes at sites removed from oriC. It is therefore a critical factor in the rescue of replication forks stalled at DNA lesions. It is also a DNA helicase. We describe insertions near the 3' end of priA that interfere with PriA activity. These insertions and the previously described priA300 encoding helicase-defective PriA K230R are shown to be effective suppressors of the DNA repair defect in recG strains, but substantially reduce the ability of ruv mutants to survive DNA damage. The data presented suggest that PriA helicase in conjunction with RecG can promote direct rescue of stalled forks independently of the recombinational pathway promoted by the combined activities of the RuvABC, RecBCD and RecA proteins, which requires only the primosome assembly activity of PriA to load DnaB at D loops. In cells lacking the helicase activity of PriA, we propose that stalled forks can be redirected to the recombination pathway via a Holliday junction intermediate common to both pathways, thus explaining the resistance of these cells to DNA damage.

Adenosine Triphosphatases↗