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At least 307 records · Page 17Linked to original sources
PubMed2005

A single domain in human DNA polymerase iota mediates interaction with PCNA: implications for translesion DNA synthesis.

DNA polymerases (Pols) of the Y family rescue stalled replication forks by promoting replication through DNA lesions. Humans have four Y family Pols, eta, iota, kappa, and Rev1, of which Pols eta, iota, and kappa have been shown to physically interact with proliferating cell nuclear antigen (PCNA) and be functionally stimulated by it. However, in sharp contrast to the large increase in processivity that PCNA binding imparts to the replicative Pol, Poldelta, the processivity of Y family Pols is not enhanced upon PCNA binding. Instead, PCNA binding improves the efficiency of nucleotide incorporation via a reduction in the apparent K(m) for the nucleotide. Here we show that Poliota interacts with PCNA via only one of its conserved PCNA binding motifs, regardless of whether PCNA is bound to DNA or not. The mode of PCNA binding by Poliota is quite unlike that in Poldelta, where multisite interactions with PCNA provide for a very tight binding of the replicating Pol with PCNA. We discuss the implications of these observations for the accuracy of DNA synthesis during translesion synthesis and for the process of Pol exchange at the lesion site.

Amino Acid Motifs↗
PubMed2006

The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex.

Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.

Carrier Proteins↗
PubMed2003

The bacterial RecA protein as a motor protein.

The bacterial RecA protein plays a central role in the repair of stalled replication forks, double-strand break repair, general recombination, induction of the SOS response, and SOS mutagenesis. The major activity of RecA in DNA metabolism is the promotion of DNA strand exchange reactions. RecA is the prototype for a ubiquitous family of proteins but exhibits a few activities that some of its eukaryotic, archaeal, and viral homologs appear to lack. In particular, the bacterial RecA protein possesses an apparent motor function that is not evident in the reactions promoted by the eukaryotic Rad51 protein. This motor may be needed only in a subset of the DNA metabolism contexts in which RecA protein functions. Models for the coupling of DNA strand exchange to ATP hydrolysis are examined.

DNA Replication↗
PubMed2005

Degradation of human exonuclease 1b upon DNA synthesis inhibition.

In response to DNA damage, signaling pathways are triggered that either block the cell division cycle at defined transitions (G1-S and G2-M) or slow down progression through the S phase. Nucleases play important roles in DNA synthesis, recombination, repair, and apoptosis. In this study, we have examined the regulation of human exonuclease 1 (hEXO1b). The endogenous hEXO1b protein was only detected upon enrichment by immunoprecipitation. We found that hEXO1b was constantly expressed throughout the cell cycle. However, treatment of cells with agents that cause arrest of DNA replication led to rapid degradation of hEXO1b. This effect was fully reversed upon removal of the block. Analysis of synchronized cells showed that degradation of hEXO1b during the S phase was strictly dependent on DNA synthesis inhibition. DNA damage caused by UV-C radiation, ionizing radiation, cisplatin, or the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine did not affect hEXO1b stability. We show that hEXO1b was phosphorylated in response to inhibition of DNA synthesis and that phosphorylation coincided with rapid protein degradation through ubiquitin-proteasome pathways. Our data support the evidence that control of exonuclease 1 activity may be critical for the maintenance of stalled replication forks.

Antibodies↗
PubMed2005

Hypoxia-induced phosphorylation of Chk2 in an ataxia telangiectasia mutated-dependent manner.

Chk2 is a serine/threonine kinase that signals to cell cycle arrest, DNA repair, and apoptotic pathways following DNA damage. It is activated by phosphorylation in response to ionizing radiation, UV light, stalled replication forks, and other types of DNA damage. Hypoxia is a common feature of solid tumors and has been shown to affect the regulation of many genes, including several DNA repair factors. We show here that Chk2 is phosphorylated on Thr68 and thereby activated in cells in response to hypoxia, and that this phosphorylation is dependent on the damage response kinase ataxia telangiectasia mutated (ATM) but not on the related kinase ATM and Rad3-related. Moreover, phosphorylation of Chk2 under hypoxia was attenuated in cells deficient in the repair factors MLH1 or NBS1. Finally, Chk2 serves to protect cells from apoptosis under hypoxic growth conditions. These results identify hypoxia as a new stimulus for Chk2 activation in an ATM-, MLH1-, and NBS1-dependent manner, and they suggest a novel pathway by which tumor hypoxia may influence cell survival and DNA repair.

Apoptosis↗
PubMed2006

Chk1- and claspin-dependent but ATR/ATM- and Rad17-independent DNA replication checkpoint response in HeLa cells.

When DNA synthesis is inhibited, DNA replication checkpoint is activated to prevent mitosis entry without fully replicated DNA. In Xenopus, caffeine-sensitive kinases [ataxia telangiectasia mutated (ATM) and ATM-related protein (ATR)] are essential in this checkpoint response, but in mammal cells an ATR/ATM-independent checkpoint response to DNA synthesis inhibition exists. Using HeLa cells, which have a caffeine-insensitive checkpoint response, we have analyzed here which molecules known to be involved in the DNA replication checkpoint participate in the caffeine-insensitive response. When DNA synthesis was inhibited in the presence of UCN01 or after knocking down Chk1 expression [Chk1 small interfering RNA (siRNA)], HeLa cells entered into aberrant mitosis. Consequently, Chk1 is essential for both the ATR/ATM-dependent and ATR/ATM-independent checkpoint response in HeLa cells. Neither wortmannin, Ly294002, nor SB202190 abrogated the caffeine-insensitive checkpoint response, indicating that DNA-PK and p38 alpha,beta are not involved in the ATR/ATM-independent Chk1 activation upon DNA synthesis inhibition. Using siRNA to knock down Rad17 and claspin, two molecules involved in sensing stalled replication forks, we also showed that claspin but not Rad17 is essential for the ATR/ATM-independent checkpoint response. Inhibition of DNA synthesis in HeLa cells led to a decrease in cyclin B1 protein accumulation that was abrogated when UCN01 was added or when claspin was knocked down. We conclude that upon DNA synthesis inhibition, Chk1 can be activated in a claspin-dependent manner independently of ATR and ATM, leading to cyclin B1 down-regulation and providing the cells of an additional mechanism to inhibit mitosis entry.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Activation of the Fanconi anemia/BRCA pathway and recombination repair in the cellular response to solar ultraviolet light.

Recombination repair plays an important role in the processing of DNA double-strand breaks (DSB) and DNA cross-links, and has been suggested to be mediated by the activation of the Fanconi anemia (FA)/BRCA pathway. Unlike DNA damage generated by ionizing radiation or DNA cross-linking, UV light-induced DNA damage is not commonly thought to require recombination for processing, as UV light does not directly induce DSBs or DNA cross-links. To elucidate the role of recombination repair in the cellular response to UV, we studied the FA/BRCA pathway in primary skin cells exposed to solar-simulated light. UV-induced monoubiquitination of the FANCD2 protein and formation of FANCD2 nuclear foci confirmed the activation of the pathway by UV light. This was only observed when cells were irradiated during S phase and was not caused by directly UV-induced DSBs. UV-exposed cells did not exhibit FANCD2 nuclear foci once they entered mitosis or when growth-arrested. In addition, UV-induced nuclear foci of the recombination proteins, RAD51 and BRCA1, colocalized with FANCD2 foci. We suggest that in response to UV light, when nucleotide excision repair failed to repair, or when translesional DNA synthesis failed to bypass UV-induced DNA photoproducts, the FA/BRCA pathway mediates the recombination repair of replication forks stalled at DNA photoproducts as a third line of defense.

BRCA1 Protein↗
PubMed2006

Radiosensitization by the poly(ADP-ribose) polymerase inhibitor 4-amino-1,8-naphthalimide is specific of the S phase of the cell cycle and involves arrest of DNA synthesis.

Radiosensitization caused by the poly(ADP-ribose) polymerase (PARP) inhibitor 4-amino-1,8-naphthalimide (ANI) was investigated in 10 asynchronously growing rodent (V79, CHO-Xrs6, CHO-K1, PARP-1+/+ 3T3, and PARP-1-/- 3T3) or human (HeLa, MRC5VI, IMR90, M059J, and M059K) cell lines, either repair proficient or defective in DNA-PK (CHO-Xrs6 and M059J) or PARP-1 (PARP-1-/- 3T3). Pulse exposure to ANI (1-hour contact) potentiated radiation response in rodent cells except in PARP-1(-/-) 3T3 fibroblasts. In contrast, ANI did not significantly enhance radiation susceptibility in asynchronously dividing human cells; yet, single-strand break rejoining was lengthened by ca. 7-fold in all but mouse PARP-1-/- 3T3s. Circumstantial evidence suggested that radiosensitization by ANI occurs in rapidly dividing cells only. Experiments using synchronized HeLa cells consistently showed that ANI-induced radiosensitization is specific of the S phase of the cell cycle and involves stalled replication forks. Under these conditions, prolonged contact with ANI ended in the formation of de novo DNA double-strand breaks hours after irradiation, evoking collision with uncontrolled replication forks of DNA lesions whose repair was impaired by inhibition of the PARP catalytic activity. The data suggest that increased response to radiotherapy by PARP inhibitors may be achieved only in rapidly growing tumors with a high S-phase content.

1-Naphthylamine↗
PubMed2004

Recombination repair pathway in the maintenance of chromosomal integrity against DNA interstrand crosslinks.

DNA interstrand crosslinks (ICL) present a major threat to cell viability and genome integrity. In eukaryotic cells, the ICLs have been suggested to be repaired by a complex process involving Xpf/Ercc1-mediated endonucleolytic incision and homologous recombination (HR). However, the entire feature of the ICL tolerating mechanism is still poorly understood. Here we studied chromosome aberrations (CA) and sister chromatid exchanges (SCE) by the use of the crosslinking agent mitomycin C (MMC), in chicken DT40 cells with the HR genes disrupted by targeted replacement. The disruption of the Rad54, Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3 genes resulted in a dramatic reduction of spontaneous and MMC-induced SCEs. Interestingly, while HR-deficient cells were hypersensitive to cell killing by MMC, MMC-induced CAs were also suppressed in the HR-deficient cells except for Rad51D-, Xrcc2- and Xrcc3-deficient cells. These observations indicate that DNA double strand breaks (DSB) at stalled replication forks and those arising as repair intermediates present strong signals to cell death but can be tolerated by the HR repair pathway, where Rad54, Rad51B and Rad51C have an initiative role and repair can be completed by their paralogs Rad51D, Xrcc2 and Xrcc3. The impairment of the HR pathway, which otherwise leads to cell death, may be somewhat substituted by an alternative mechanism such as the Mre11/Rad50/Nbs1 pathway, resulting in reduced frequencies of SCEs and CAs.

Animals↗
PubMed2004

Mechanisms and consequences of methylating agent-induced SCEs and chromosomal aberrations: a long road traveled and still a far way to go.

Since the milestone work of Evans and Scott, demonstrating the replication dependence of alkylation-induced aberrations, and Obe and Natarajan, pointing to the critical role of DNA double-strand breaks (DSBs) as the ultimate trigger of aberrations, the field has grown extensively. A notable example is the identification of DNA methylation lesions provoking chromosome breakage (clastogenic) effects, which made it possible to model clastogenic pathways evoked by genotoxins. Experiments with repair-deficient mutants and transgenic cell lines revealed both O6-methylguanine (O6MeG) and N- methylpurines as critical lesions. For S(N)2 agents such as methyl- methanesulfonate (MMS), base N-methylation lesions are most critical, likely because of the formation of apurinic sites blocking replication. For S(N)1 agents, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O6-methylguanine (O6MeG) plays the major role both in recombination and clastogenicity in the post-treatment cell cycle, provided the lesion is not pre-replicatively repaired by O6-methylguanine-DNA methyltransferase (MGMT). The conversion probability of O6MeG into SCEs and chromosomal aberrations is estimated to be about 30:1 and >10,000:1 respectively, indicating this mispairing pro-mutagenic lesion to be highly potent in inducing recombination giving rise to SCEs. O6MeG needs replication and mismatch repair to become converted into a critical secondary genotoxic lesion. Here it is proposed that this secondary lesion can be tolerated by a process termed recombination bypass. This process is supposed to be important in the tolerance of lesions that can not be processed by translesion synthesis accomplished by low-fidelity DNA polymerases. Recombination bypass results in SCEs and might represent an alternative pathway of tolerance of non-instructive lesions. In the case of O6MeG-derived secondary lesions, recombination bypass appears to protect against cell killing since SCEs are already induced with low, non-toxic doses of MNNG. Saturation of lesion tolerance by recombination bypass or translesion synthesis may cause block of DNA replication leading to DSBs at stalled replication forks, which result in chromatid-type aberrations. Along with this model, several putative consequences of methylation-induced aberrations will be discussed such as cell death by apoptosis as well its role in tumor promotion and progression.

Alkylating Agents↗
PubMed2006

The fission yeast DNA structure checkpoint protein Rad26ATRIP/LCD1/UVSD accumulates in the cytoplasm following microtubule destabilization.

BACKGROUND: DNA structure checkpoints are conserved eukaryotic signal transduction pathways that help preserve genomic integrity. Upon detecting checkpoint signals such as stalled replication forks or double-stranded DNA breaks, these pathways coordinate appropriate stress responses. Members of the PI-3 kinase related kinase (PIKK) family are essential elements of DNA structure checkpoints. In fission yeast, the Rad3 PIKK and its regulatory subunit Rad26 coordinate the detection of checkpoint signals with pathway outputs. RESULTS: We found that untreated rad26Delta cells were defective for two microtubule-dependent processes: chromosome segregation and morphogenesis. Interestingly, cytoplasmic accumulation of Rad26-GFP occurred following treatment with microtubule destabilizing drugs, but not during treatment with the genotoxic agent Phleomycin. Cytoplasmic accumulation of Rad26-GFP depended on Rad24, a 14-3-3 protein also required for DNA structure checkpoints and morphogenesis. Results of over expression and epistasis experiments confirm that Rad26 and Rad24 define a response to microtubule destabilizing conditions. CONCLUSION: Two DNA structure checkpoint proteins with roles in morphogenesis define a response to microtubule destabilizing conditions.

Actins↗
PubMed2005

UV irradiation induces homologous recombination genes in the model archaeon, Halobacterium sp. NRC-1.

BACKGROUND: A variety of strategies for survival of UV irradiation are used by cells, ranging from repair of UV-damaged DNA, cell cycle arrest, tolerance of unrepaired UV photoproducts, and shielding from UV light. Some of these responses involve UV-inducible genes, including the SOS response in bacteria and an array of genes in eukaryotes. To address the mechanisms used in the third branch of life, we have studied the model archaeon, Halobacterium sp. strain NRC-1, which tolerates high levels of solar radiation in its natural hypersaline environment. RESULTS: Cells were irradiated with 30-70 J/m(2) UV-C and an immunoassay showed that the resulting DNA damage was largely repaired within 3 hours in the dark. Under such conditions, transcriptional profiling showed the most strongly up-regulated gene was radA1, the archaeal homolog of rad51/recA, which was induced 7-fold. Additional genes involved in homologous recombination, such as arj1 (recJ-like exonuclease), dbp (eukaryote-like DNA binding protein of the superfamily I DNA and RNA helicases), and rfa3 (replication protein A complex), as well as nrdJ, encoding for cobalamin-dependent ribonucleotide reductase involved in DNA metabolism, was also significantly induced in one or more of our experimental conditions. Neither prokaryotic nor eukaryotic excision repair gene homologs were induced and there was no evidence of an SOS-like response. CONCLUSION: These results show that homologous recombination plays an important role in the cellular response of Halobacterium sp. NRC-1 to UV damage. Homologous recombination may permit rescue of stalled replication forks, and/or facilitate recombinational repair. In either case, this provides a mechanism for the observed high-frequency recombination among natural populations of halophilic archaea.

Journal Article↗
PubMed2006

"ATR activation in response to ionizing radiation: still ATM territory".

Unrepaired DNA double-strand breaks (DSBs) are a major cause for genomic instability. Therefore, upon detection of a DSB a rapid response must be assembled to coordinate the proper repair/signaling of the lesion or the elimination of cells with unsustainable amounts of DNA damage. Three members of the PIKK family of protein kinases -ATM, ATR and DNA-PKcs- take the lead and initiate the signaling cascade emanating from DSB sites. Whereas DNA-PKcs activity seems to be restricted to the phosphorylation of targets involved in DNA repair, ATM and ATR phosphorylate a broad spectrum of cell cycle regulators and DNA repair proteins. In the canonical model, ATM and ATR are activated by two different types of lesions and signal through two independent and alternate pathways. Specifically, ATR is activated by various forms of DNA damage, including DSBs, arising at stalled replication forks ("replication stress"), and ATM is responsible for the signaling of DSBs that are not associated with the replication machinery throughout the cell cycle. Recent evidence suggests that this model might be oversimplified and that coordinated crosstalk between ATM and ATR activation routes goes on at the core of the DNA damage response.

Journal Article↗
PubMed2005

A role for the fission yeast Rqh1 helicase in chromosome segregation.

Schizosaccharomyces pombe Rqh1 protein is a member of the RecQ DNA helicase family. Members of this protein family are mutated in several human genome instability syndromes, including Bloom, Werner and Rothmund-Thomson syndromes. RecQ helicases participate in recombination repair of stalled replication forks or DNA breaks, but the precise mechanisms that lead to the development of cancer in these diseases have remained obscure. Here, we reveal a function for Rqh1 in chromosome segregation even in the absence of exogenous insult to the DNA. We show that cells lacking Rqh1 are delayed in anaphase progression, and show lagging chromosomal DNA, which is particularly apparent in the rDNA locus. This mitotic delay is dependent on the spindle checkpoint, as deletion of mad2 abolishes the delay as well as the accumulation of Cut2 in rqh1delta cells. Furthermore, relieving replication fork arrest in the rDNA repeat by deletion of reb1+ partially suppresses rqh1delta phenotypes. These data are consistent with the function of the Top3-RecQ complex in maintenance of the rDNA structure by processing aberrant chromosome structures arising from DNA replication. The chromosome segregation defects seen in the absence of functional RecQ helicases may contribute to the pathogenesis of human RecQ helicase disorders.

Abnormalities, Multiple↗
PubMed2006

Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length.

In Saccharomyces cerevisiae, DNA replication stress activates the replication checkpoint, which slows S-phase progression, stabilizes slowed or stalled replication forks, and relieves inhibition of the ribonucleotide reductase (RNR) complex. To identify novel genes that promote cellular viability after replication stress, the S. cerevisiae non-essential haploid gene deletion set (4812 strains) was screened for sensitivity to the RNR inhibitor hydroxyurea (HU). Strains bearing deletions in either CCR4 or CAF1/POP2, which encode components of the cytoplasmic mRNA deadenylase complex, were particularly sensitive to HU. We found that Ccr4 cooperated with the Dun1 branch of the replication checkpoint, such that ccr4Delta dun1Delta strains exhibited irreversible hypersensitivity to HU and persistent activation of Rad53. Moreover, because ccr4Delta and chk1Delta exhibited epistasis in several genetic contexts, we infer that Ccr4 and Chk1 act in the same pathway to overcome replication stress. A counterscreen for suppressors of ccr4Delta HU sensitivity uncovered mutations in CRT1, which encodes the transcriptional repressor of the DNA-damage-induced gene regulon. Whereas Dun1 is known to inhibit Crt1 repressor activity, we found that Ccr4 regulates CRT1 mRNA poly(A) tail length and may subtly influence Crt1 protein abundance. Simultaneous overexpression of RNR2, RNR3 and RNR4 partially rescued the HU hypersensitivity of a ccr4Delta dun1Delta strain, consistent with the notion that the RNR genes are key targets of Crt1. These results implicate the coordinated regulation of Crt1 via Ccr4 and Dun1 as a crucial nodal point in the response to DNA replication stress.

Cell Cycle Proteins↗
PubMed2006

Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest.

Werner syndrome (WS) is a rare genetic disorder characterized by genomic instability caused by defects in the WRN gene encoding a member of the human RecQ helicase family. RecQ helicases are involved in several DNA metabolic pathways including homologous recombination (HR) processes during repair of stalled replication forks. Following introduction of interstrand DNA crosslinks (ICL), WRN relocated from nucleoli to arrested replication forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, we use fluorescence resonance energy transfer (FRET) and immune-precipitation experiments to demonstrate that WRN participates in a multiprotein complex including RAD51, RAD54, RAD54B and ATR in cells where replication has been arrested by ICL. We verify the WRN-RAD51 and WRN-RAD54B direct interaction in vitro. Our data support a role for WRN also in the recombination step of ICL repair.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2005

Multiple endonucleases function to repair covalent topoisomerase I complexes in Saccharomyces cerevisiae.

Topoisomerase I plays a vital role in relieving tension on DNA strands generated during replication. However if trapped by camptothecin or other DNA damage, topoisomerase protein complexes may stall replication forks producing DNA double-strand breaks (DSBs). Previous work has demonstrated that two structure-specific nucleases, Rad1 and Mus81, protect cells from camptothecin toxicity. In this study, we used a yeast deletion pool to identify genes that are important for growth in the presence of camptothecin. In addition to genes involved in DSB repair and recombination, we identified four genes with known or implicated nuclease activity, SLX1, SLX4, SAE2, and RAD27, that were also important for protection against camptothecin. Genetic analysis revealed that the flap endonucleases Slx4 and Sae2 represent new pathways parallel to Tdp1, Rad1, and Mus81 that protect cells from camptothecin toxicity. We show further that the function of Sae2 is likely due to its interaction with the endonuclease Mre11 and that the latter acts on an independent branch to repair camptothecin-induced damage. These results suggest that Mre11 (with Sae2) and Slx4 represent two new structure-specific endonucleases that protect cells from trapped topoisomerase by removing topoisomerase-DNA adducts.

Camptothecin↗
PubMed2006

Mechanisms of eukaryotic DNA double strand break repair.

For all cells, a DNA double strand break (DSB) is a dangerous lesion that can have profound consequences for the genome. If a DSB is encountered during mitosis, chromosomal separation may be adversely affected. Alternatively, during S phase a DSB may cause replication fork stalling or collapse. Improperly repaired DSBs can result in chromosomal rearrangements, senescence or activation of apoptotic pathways. Cells have developed sophisticated recombination pathways to metabolize and repair DSBs quickly as well as the capacity to differentiate physiologically occurring breaks from life threatening lesions. The two major pathways of recombination repair are known as non-homologous end-joining (NHEJ) and homologous recombination (HR). In this review, we will discuss the detection, response, and repair of DSBs in eukaryotes.

Antigens, Nuclear↗