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PubMed1983

Do stalled replication forks synthesize a specific alarmone?

Potential causes of premature arrest of a replication fork in vivo include an encounter with a chemical lesion in the DNA, inhibition of one of the essential enzymes of the fork, and spontaneous failure of the fork due to its finite degree of processivity. I suggest that a premature arrest of either a eukaryotic or prokaryotic replication fork induces it to enter a different state in which the fork synthesizes a specific signal nucleotide ("alarmone"). One function of the postulated new alarmone would be to increase the probability of re-initiation of DNA replication, either in cis (at an origin proximal to a site of the fork arrest) or in trans (at many different origins). An additional, mechanistically related function of the postulated alarmone could be to increase the probability of re-assembly of an arrested fork beyond an otherwise impassable DNA lesion. In case of multiple fork arrests, an alarmone-mediated increase in the probability of replicon reinitiation (disproportionate DNA replication) would result in gene amplification at many different loci, thereby increasing the probability of cell's survival in a cytotoxic medium. Other likely functions of a fork-produced alarmone may include stimulation of DNA repair pathways including excision repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed2005

Sumoylation of PCNA: Wrestling with recombination at stalled replication forks.

Post-replication repair encompassses error-prone and error-free processes for bypassing lesions encountered during DNA replication. In Saccharomyces cerevisiae, proteins acting in the Rad6-dependent pathway are required to channel lesions into these pathways. Until recently there was little information as to how this channelling was regulated. However, several recent papers, and in particular from the Jentsch and Ulrich groups have provided striking insights into the role of modified forms of PCNA in these events [C. Hoege, B. Pfander, G.L. Moldovan, G. Pyrowolakis, S. Jentsch, RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO, Nature 419 (2002) 135-141; P. Stelter, H.D. Ulrich, Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation, Nature 425 (2003) 188-191; B. Pfander, G.L. Moldovan, M. Sacher, C. Hoege, S. Jentsch, SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase, Nature 436 (2005) 428-433; E. Papouli, S. Chen, A.A. Davies, D. Huttner, L. Krejci, P. Sung, H.D. Ulrich, Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p, Mol. Cell. 19 (2005) 123-133]. In particular they have shown that mono-ubiquitinated PCNA directs translesion synthesis via DNA polymerases with low stringency, and that polyubiquitinated PCNA is associated with error-free avoidance of lesions. Recent data have shown that the role of small ubiquitin-like modifier (SUMO) modification of PCNA is not an event that occurs merely in the absence of ubiquitination, rather it serves to recruit Srs2 to replication forks in order to inhibit recombination. The implications of these findings for post-replication repair in S. cerevisiae and other eukaryotes are discussed.

Animals↗
PubMed2000

The importance of repairing stalled replication forks.

The bacterial SOS response to unusual levels of DNA damage has been recognized and studied for several decades. Pathways for re-establishing inactivated replication forks under normal growth conditions have received far less attention. In bacteria growing aerobically in the absence of SOS-inducing conditions, many replication forks encounter DNA damage, leading to inactivation. The pathways for fork reactivation involve the homologous recombination systems, are nonmutagenic, and integrate almost every aspect of DNA metabolism. On a frequency-of-use basis, these pathways represent the main function of bacterial DNA recombination systems, as well as the main function of a number of other enzymatic systems that are associated with replication and site-specific recombination.

Bacteria↗
PubMed2001

The DNA replication checkpoint response stabilizes stalled replication forks.

In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase. Active Rad53 slows DNA synthesis when DNA is damaged and prevents firing of late origins of replication. Further, rad53 mutants are unable to recover from a replication block. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes. Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.

Cell Cycle↗
PubMed2004

Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks.

In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or lambda-phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction.

Acid Anhydride Hydrolases↗
PubMed2005

Stabilization of a stalled replication fork by concerted actions of two helicases.

PriA helicase plays crucial roles in restoration of arrested replication forks. It carries a "3' terminus binding pocket" in its N-terminal DNA binding domain, which is required for high affinity binding of PriA to a fork carrying a 3'-end of a nascent leading strand at the branch. We show that the abrogation of the 3' terminus recognition either by a mutation in the 3' terminus binding pocket or by the bulky modification of the 3'-end leads to unwinding of the unreplicated duplex arm on this fork, causing potential fork destabilization. This indicates a critical role of the 3' terminus binding pocket of PriA in its "stable" binding at the fork for primosome assembly. In contrast, PriA unwinds the unreplicated duplex region on a fork without a 3'-end, potentially destabilizing the fork. However, this process is inhibited by RecG helicase, capable of regressing the fork until the 3'-end of the nascent leading strand reaches the branch. PriA now stably binds to this regressed fork, stabilizing it. Using a model arrest-fork-substrate, we reconstitute the above process in vitro with RecG and PriA proteins. Our results present a novel mechanism by which two helicases function in a highly coordinated manner to generate a structure in which an arrested fork is stabilized for further repair and/or replication restart.

Adenosine Triphosphatases↗
PubMed2005

Dimerization of the ATRIP protein through the coiled-coil motif and its implication to the maintenance of stalled replication forks.

ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Deletions at stalled replication forks occur by two different pathways.

Replication blockage induces non-homologous deletions in Escherichia coli. The mechanism of the formation of these deletions was investigated. A pBR322-mini-oriC hybrid plasmid carrying two E. coli replication terminators (Ter sites) in opposite orientations was used. Deletions which remove at least the pBR322 blocking site (named Ter1) occurred at a frequency of 2 x 10(-6) per generation. They fall into two equally large classes: deletions that join sequences with no homology, and others that join sequences of 3-10 bp of homology. Some 95% of the deletions in the former class resulted from the fusion of sequences immediately preceding the two Ter sites, indicating a direct role for blocked replication forks in their formation. These deletions were not found in a topA10 mutant, suggesting a topoisomerase I-mediated process. In contrast, deletions joining short homologous sequences were not affected by the topA10 mutation. However, the incidence of this second class of deletions increased 10-fold in a recD mutant, devoid of exonuclease V activity. This indicates that linear molecules are intermediates in their formation. In addition, approximately 50% of these deletions were clustered in the region flanking the Ter1 site. We propose that they are produced by repair of molecules broken at the blocked replication forks.

Base Sequence↗
PubMed2005

Viral transport of DNA damage that mimics a stalled replication fork.

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.

Carrier Proteins↗
PubMed2002

Direct rescue of stalled DNA replication forks via the combined action of PriA and RecG helicase activities.

The PriA protein of Escherichia coli plays a key role in the rescue of replication forks stalled on the template DNA. One attractive model of rescue relies on homologous recombination to establish a new fork via PriA-mediated loading of the DnaB replicative helicase at D loop intermediates. We provide genetic and biochemical evidence that PriA helicase activity can also rescue a stalled fork by an alternative mechanism that requires manipulation of the fork before loading of DnaB on the lagging strand template. This direct rescue depends on RecG, which unwinds forks and Holliday junctions and interconverts these structures. The combined action of PriA and RecG helicase activities may thus avoid the potential dangers of rescue pathways involving fork breakage and recombination.

Adenosine Triphosphatases↗
PubMed2004

DNA replication: stalling a fork for imprinting and switching.

Mating-type switching in fission yeast has long been known to be directed by a DNA 'imprint'. This imprint has now been firmly characterized as a protected site-specific and strand-specific nick. New work also links the widely conserved Swi1-Swi3 complex to the protection of stalled replication forks in general.

Cell Cycle Proteins↗
PubMed2003

Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis.

Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polkappa). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.

Cell Cycle↗
PubMed2005

Suppression of genomic instability by SLX5 and SLX8 in Saccharomyces cerevisiae.

Replication forks can stall spontaneously at specific sites in the genome, and upon encountering DNA lesions resulting from chemical or radiation damage. In Saccharomyces cerevisiae proteins implicated in processing of stalled replication forks include those encoded by the SGS1, TOP3, MUS81, MMS4, SLX1, SLX4, SLX5/HEX3, and SLX8 genes. We tested the roles of these genes in suppressing gross chromosomal rearrangements (GCRs), which include translocations, large interstitial deletions, and loss of a chromosome arm with de novo telomere addition. We found that mus81, mms4, slx1, slx4, slx5, and slx8 mutants all have elevated levels of spontaneous GCRs, and that SLX5 and SLX8 are particularly critical suppressors of GCRs during normal cell cycle progression. In addition to increased GCRs, deletion of SLX5 or SLX8 resulted in increased relocalization of the DNA damage checkpoint protein Ddc2 and activation of the checkpoint kinase Rad53, indicating the accumulation of spontaneous DNA damage. Surprisingly, mutants in slx5 or slx8 were not sensitive to transient replication fork stalling induced by hydroxyurea, nor were they sensitive to replication dependent double-strand breaks induced by camptothecin. This suggested that Slx8 and Slx8 played limited roles in stabilizing, restarting, or resolving transiently stalled replication forks, but were critical for preventing the accumulation of DNA damage during normal cell cycle progression.

Cell Cycle Proteins↗
PubMed2002

Mus81-Eme1 and Rqh1 involvement in processing stalled and collapsed replication forks.

The processing of stalled replication forks and the repair of collapsed replication forks are essential functions in all organisms. In fission yeast DNA junctions at stalled replication forks appear to be processed by either the Rqh1 DNA helicase or Mus81-Eme1 endonuclease. Accordingly, we show that the hypersensitivity to agents that cause replication fork stalling of mus81, eme1, and rqh1 mutants is suppressed by a Holliday junction resolvase (RusA), as is the synthetic lethality of a mus81(-) rqh1(-) double mutant. Recombinant Mus81-Eme1, purified from Escherichia coli, readily cleaves replication fork structures but cleaves synthetic Holliday junctions relatively poorly in vitro. From these data we propose that Mus81-Eme1 can process stalled replication forks before they have regressed to form a Holliday junction. We also implicate Mus81-Eme1 and Rqh1 in the repair of collapsed replication forks. Here Mus81-Eme1 and Rqh1 seem to function on different substrates because RusA can substitute for Mus81-Eme1 but not Rqh1.

Camptothecin↗
PubMed2003

Identification and characterization of the human mus81-eme1 endonuclease.

The faithful and complete replication of DNA is necessary for the maintenance of genome stability. It is known, however, that replication forks stall at lesions in the DNA template and need to be processed so that replication restart can occur. In fission yeast, the Mus81-Eme1 endonuclease complex (Mus81-Mms4 in Saccharomyces cerevisiae) has been implicated in the processing of aberrant replication intermediates. In this report, we identify the human homolog of the Schizosaccharomyces pombe EME1 gene and have purified the human Mus81-Eme1 heterodimer. We show that Mus81-Eme1 is an endonuclease that exhibits a high specificity for synthetic replication fork structures and 3'-flaps in vitro. The nuclease cleaves Holliday junctions inefficiently ( approximately 75-fold less than flap or fork structures), although cleavage can be increased 6-fold by the presence of homologous sequences previously shown to permit base pair "breathing." We conclude that human Mus81-Eme1 is a flap/fork endonuclease that is likely to play a role in the processing of stalled replication fork intermediates.

Amino Acid Sequence↗
PubMed2005

Schizosaccharomyces pombe Swi1, Swi3, and Hsk1 are components of a novel S-phase response pathway to alkylation damage.

The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1. This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage. Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.

Alkylation↗
PubMed2004

Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance.

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.

Cell Cycle↗