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PubMed2006

Genetic instability in cancer cells by impaired cell cycle checkpoints.

Cells continuously encounter DNA damage caused either by damaging agents, including oxygen radicals and DNA replication errors caused by stalled replication forks, or by extracellular environments such as ultraviolet or ionizing irradiation. Such DNA damage poses a great threat to genome stability, potentially leading to loss or amplification of chromosome activity, which may result in cellular senescence, cancer or apoptosis. The DNA damage checkpoints coordinate an arrest in cell cycle progression with the DNA repair process, suppressing either mitotic catastrophe or proliferation of cells with damaged DNA. Numerous key players have been identified in terms of damage sensor proteins, transducer kinases and effectors, but their coordination and interconnectedness in damage control have only recently become evident. In this review, we discuss changes in chromatin structure, recruitment of mediator proteins and activation of transducer kinases in response to DNA damage. These cellular responses are important for determining the potential effects of current cancer therapies in terms of toxicity and efficacy.

Cell Cycle↗

PubMed2006

The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activation of Chk1.

ATR-dependent activation of the kinase Chk1 is the initial step in signal transduction in the DNA replication checkpoint, which allows a cell to enter mitosis only after the completion of DNA replication. TopBP1-related proteins in higher eukaryotes are implicated in the replication checkpoint, but their exact role remains elusive because of their requirements for replication initiation. Here we report that the initiation function of Xenopus Cut5/TopBP1 could be entirely separated from its checkpoint function: the N-terminal half fragment, a region of Cut5 conserved through evolution, is sufficient for initiation, but is incapable of activating the checkpoint; the C-terminal half fragment, which is unique in metazoan species, is by itself capable of activating the checkpoint response without initiating replication. Upon the activation of Chk1, the Ser1131 within the C-terminal region of Cut5 is phosphorylated, and this phosphorylation is critical for the checkpoint response. Furthermore, Cut5 directly stimulated Chk1 phosphorylation in the in vitro kinase assay reconstituted with recombinant proteins and ATR immunoprecipitated from extracts. On the basis of replication protein A (RPA)-dependent loading of Cut5 on to replicating and replication-arrested chromatin, we propose that Cut5 plays a crucial role in the initial amplification step of the ATR-Chk1 signaling pathway at the stalled replication fork.

Animals↗

PubMed2004

Control of DNA replication initiation by recruitment of an essential initiation protein to the membrane of Bacillus subtilis.

The Bacillus subtilis proteins DnaD and DnaB are essential for replication initiation and are conserved in low G+C content Gram-positive bacteria. Previous work indicated that DnaD and DnaB are involved in helicase loading during the process of restarting stalled replication forks. We have investigated the roles of DnaD and DnaB in replication initiation at oriC in vivo. Using chromatin immunoprecipitation (ChIP), we found that DnaD and DnaB functions are needed to load the replicative helicase at oriC. To investigate further the functions of DnaD and DnaB in replication initiation, we isolated and characterized suppressors of the temperature sensitivity of dnaD and dnaB mutant cells. In both cases, we isolated the identical missense mutation in dnaB, dnaBS371P. Using yeast two-hybrid analysis, we found that dnaBS371P uncovers a previously undetected physical interaction between DnaD and DnaB. We also found that DnaBS371P constitutively recruits DnaD to the membrane fraction of cells, where DnaB and oriC are enriched. Phenotypes of cells expressing DnaBS371P are consistent with aberrant replication control. We hypothesize that B. subtilis regulates replication initiation by regulating a physical interaction between two proteins essential for helicase loading at chromosomal origins.

Bacillus subtilis↗

PubMed2004

IS911 partial transposition products and their processing by the Escherichia coli RecG helicase.

Insertion of bacterial insertion sequence IS911 can often be directed to sequences resembling its ends. We have investigated this type of transposition and shown that it can occur via cleavage of a single end and its targeted transfer next to another end. The single end transfer (SET) events generate branched DNA molecules that contain a nicked Holliday junction and can be considered as partial transposition products. Our results indicate that these can be processed by the Escherichia coli host independently of IS911-encoded proteins. Such resolution depends on the presence of homologous DNA regions neighbouring the cross-over point in the SET molecule. Processing is often accompanied by sequence conversion between donor and target sequences, suggesting that branch migration is involved. We show that resolution is greatly reduced in a recG host. Thus, the branched DNA-specific helicase, RecG, involved in processing of potentially lethal DNA structures such as stalled replication forks, also intervenes in the resolution of partial IS911 transposition products.

Base Sequence↗

PubMed2005

Host factors that promote transpososome disassembly and the PriA-PriC pathway for restart primosome assembly.

Initiation of bacteriophage Mu DNA replication by transposition requires the disassembly of the transpososome that catalyses strand exchange and the assembly of a replisome promoted by PriA, PriB, PriC and DnaT proteins, which function in the host to restart stalled replication forks. Once the molecular chaperone ClpX weakens the very tight binding of the transpososome to the Mu ends, host disassembly factors (MRFalpha-DF) promote the dissociation of the transpososome from the DNA template and the assembly of a new nucleoprotein complex. Prereplisome factors (MRFalpha-PR) further alter the complex, allowing PriA binding and loading of major replicative helicase DnaB onto the template promoted by the restart proteins. MRFalpha-PR is essential for DnaB loading by restart proteins even on the deproteinized Mu fork whereas MRFalpha-DF is not required on the deproteinized template. When the transition from transpososome to replisome was reconstituted using MRFalpha-DF and MRFalpha-PR, initiation of Mu DNA replication was strictly dependent upon added PriC and PriA helicase. In contrast, initiation on the deproteinized template was predominantly dependent upon PriB and did not require PriA's helicase activity. The results indicate that transition mechanisms beginning with the transpososome disassembly can determine the pathway of replisome assembly by restart proteins.

Adenosine Triphosphatases↗

PubMed2004

Involvement of mammalian Mus81 in genome integrity and tumor suppression.

Mus81-Eme1 endonuclease has been implicated in the rescue of stalled replication forks and the resolution of meiotic recombination intermediates in yeast. We used gene targeting to study the physiological requirements of Mus81 in mammals. Mus81-/- mice are viable and fertile, which indicates that mammalian Mus81 is not essential for recombination processes associated with meiosis. Mus81-deficient mice and cells were hypersensitive to the DNA cross-linking agent mitomycin C but not to gamma-irradiation. Remarkably, both homozygous Mus81-/- and heterozygous Mus81+/- mice exhibited a similar susceptibility to spontaneous chromosomal damage and a profound and equivalent predisposition to lymphomas and other cancers. These studies demonstrate a critical role for the proper biallelic expression of the mammalian Mus81 in the maintenance of genomic integrity and tumor suppression.

Alleles↗

PubMed2006

Checkpoint proteins control survival of the postmitotic cells in Caenorhabditis elegans.

Checkpoints are evolutionarily conserved signaling mechanisms that arrest cell division and alter cellular stress resistance in response to DNA damage or stalled replication forks. To study the consequences of loss of checkpoint functions in whole animals, checkpoint genes were inactivated in the nematode C. elegans. We show that checkpoint proteins are not only essential for normal development but also determine adult somatic maintenance. Checkpoint proteins play a role in the survival of postmitotic adult cells.

Adaptation, Physiological↗

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↗

PubMed2001

Growth phase variation in cell and nucleoid morphology in a Bacillus subtilis recA mutant.

The major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth, Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for addB and recF mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (approximately 15%) of abnormally long cells and nucleoids developed in B. subtilis recA mutants. In addition, recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation.

Bacillus subtilis↗

PubMed2002

The RuvABC resolvase is indispensable for recombinational repair in sbcB15 mutants of Escherichia coli.

The RuvABC proteins of Escherichia coli play an important role in the processing of Holliday junctions during homologous recombination and recombinational repair. Mutations in the ruv genes have a moderate effect on recombination and repair in wild-type strains but confer pronounced recombination deficiency and extreme sensitivity to DNA-damaging agents in a recBC sbcBC background. Genetic analysis presented in this work revealed that the (Delta)ruvABC mutation causes an identical DNA repair defect in UV-irradiated recBC sbcBC, sbcBC, and sbcB strains, indicating that the sbcB mutation alone is responsible for the extreme UV sensitivity of recBC sbcBC ruv derivatives. In experiments with gamma irradiation and in conjugational crosses, however, sbcBC (Delta)ruvABC and sbcB (Delta)ruvABC mutants displayed higher recombination proficiency than the recBC sbcBC (Delta)ruvABC strain. The frequency of conjugational recombination observed with the sbcB (Delta)ruvABC strain was quite similar to that of the (Delta)ruvABC single mutant, indicating that the sbcB mutation does not increase the requirement for RuvABC in a recombinational process starting from preexisting DNA ends. The differences between the results obtained in three experimental systems used suggest that in UV-irradiated cells, the RuvABC complex might act in an early stage of recombinational repair. The results of this work are discussed in the context of recent recombination models which propose the participation of RuvABC proteins in the processing of Holliday junctions made from stalled replication forks. We suggest that the mutant SbcB protein stabilizes these junctions and makes their processing highly dependent on RuvABC resolvase.

Bacterial Proteins↗

PubMed2006

ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress.

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2006

Functional and physical interaction of yeast Mgs1 with PCNA: impact on RAD6-dependent DNA damage tolerance.

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.

Adenosine Triphosphatases↗

PubMed2000

Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1.

Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) protein-docking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of phenotypes. Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment. Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis. We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.

Amino Acid Sequence↗

PubMed2002

A conserved domain of Schizosaccharomyces pombe dfp1(+) is uniquely required for chromosome stability following alkylation damage during S phase.

The fission yeast Dbf4 homologue Dfp1 has a well-characterized role in regulating the initiation of DNA replication. Sequence analysis of Dfp1 homologues reveals three highly conserved regions, referred to as motifs N, M, and C. To determine the roles of these conserved regions in Dfp1 function, we have generated dfp1 alleles with mutations in these regions. Mutations in motif N render cells sensitive to a broad range of DNA-damaging agents and replication inhibitors, yet these mutant proteins are efficient activators of Hsk1 kinase in vitro. In contrast, mutations in motif C confer sensitivity to the alkylating agent methyl methanesulfonate (MMS) but, surprisingly, not to UV, ionizing radiation, or hydroxyurea. Motif C mutants are poor activators of Hsk1 in vitro but can fulfill the essential function(s) of Dfp1 in vivo. Strains carrying dfp1 motif C mutants have an intact mitotic and intra-S-phase checkpoint, and epistasis analysis indicates that dfp1 motif C mutants function outside of the known MMS damage repair pathways, suggesting that the observed MMS sensitivity is due to defects in recovery from DNA damage. The motif C mutants are most sensitive to MMS during S phase and are partially suppressed by deletion of the S-phase checkpoint kinase cds1. Following treatment with MMS, dfp1 motif C mutants exhibit nuclear fragmentation, chromosome instability, precocious recombination, and persistent checkpoint activation. We propose that Dfp1 plays at least two genetically separable roles in the DNA damage response in addition to its well-characterized role in the initiation of DNA replication and that motif C plays a critical role in the response to alkylation damage, perhaps by restarting or stabilizing stalled replication forks.

Alkylation↗

PubMed2004

Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes.

The Werner and Bloom syndromes are caused by loss-of-function mutations in WRN and BLM, respectively, which encode the RecQ family DNA helicases WRN and BLM, respectively. Persons with Werner syndrome displays premature aging of the skin, vasculature, reproductive system, and bone, and those with Bloom syndrome display more limited features of aging, including premature menopause; both syndromes involve genome instability and increased cancer. The proteins participate in recombinational repair of stalled replication forks or DNA breaks, but the precise functions of the proteins that prevent rapid aging are unknown. Accumulating evidence points to telomeres as targets of WRN and BLM, but the importance in vivo of the proteins in telomere biology has not been tested. We show that Wrn and Blm mutations each accentuate pathology in later-generation mice lacking the telomerase RNA template Terc, including acceleration of phenotypes characteristic of latest-generation Terc mutants. Furthermore, pathology not observed in Terc mutants but similar to that observed in Werner syndrome and Bloom syndrome, such as bone loss, was observed. The pathology was accompanied by enhanced telomere dysfunction, including end-to-end chromosome fusions and greater loss of telomere repeat DNA compared with Terc mutants. These findings indicate that telomere dysfunction may contribute to the pathogenesis of Werner syndrome and Bloom syndrome.

Animals↗

PubMed2004

The protein kinase Snf1 is required for tolerance to the ribonucleotide reductase inhibitor hydroxyurea.

The Snf1/AMP-activated kinases are involved in a wide range of stress responses in eukaryotic cells. We discovered a novel role for the Snf1 kinase in the cellular response to genotoxic stress in yeast. snf1 mutants are hypersensitive to hydroxyurea (HU), methyl-methane sulfonate, and cadmium, but they are not sensitive to several other genotoxic agents. HU inhibits ribonucleotide reductase (RNR), and deletion of SNF1 also increased the growth defects of an rnr4 ribonucleotide reductase mutant. The snf1 mutant has a functional checkpoint response to HU insofar as cells arrest division normally and derepress the transcription of RNR genes. The sensitivity of snf1 to HU or to RNR4 deletion may be due to posttranscriptional defects in RNR function or to defects in the repair of, and recovery from, stalled replication forks. The Mig3 repressor was identified as one target of Snf1 in this pathway. Genetic and biochemical analyses suggest that a weak kinase activity is sufficient to confer resistance to HU, whereas a high level of kinase activity is required for optimal growth on carbon sources other than glucose. Quantitative regulation of Snf1 kinase activity may contribute to the specificity of the effector responses that it controls.

Cadmium↗

PubMed2005

Chk1-dependent S-M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures.

We investigated mitotic delay during replication arrest (the S-M checkpoint) in DT40 B-lymphoma cells deficient in the Chk1 or Chk2 kinase. We show here that cells lacking Chk1, but not those lacking Chk2, enter mitosis with incompletely replicated DNA when DNA synthesis is blocked, but only after an initial delay. This initial delay persists when S-M checkpoint failure is induced in Chk2-/- cells with the Chk1 inhibitor UCN-01, indicating that it does not depend on Chk1 or Chk2 activity. Surprisingly, dephosphorylation of tyrosine 15 did not accompany Cdc2 activation during premature entry to mitosis in Chk1-/- cells, although mitotic phosphorylation of cyclin B2 did occur. Previous studies have shown that Chk1 is required to stabilize stalled replication forks during replication arrest, and strikingly, premature mitosis occurs only in Chk1-deficient cells which have lost the capacity to synthesize DNA as a result of progressive replication fork inactivation. These results suggest that Chk1 maintains the S-M checkpoint indirectly by preserving the viability of replication structures and that it is the continued presence of such structures, rather than the activation of Chk1 per se, which delays mitosis until DNA replication is complete.

Animals↗

PubMed2005

Nuclear dynamics of PCNA in DNA replication and repair.

The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a "sliding clamp" that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.

Animals↗