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PubMed2006

Mgs1 and Rad18/Rad5/Mms2 are required for survival of Saccharomyces cerevisiae mutants with novel temperature/cold sensitive alleles of the DNA polymerase delta subunit, Pol31.

Saccharomyces cerevisiae DNA polymerase delta (Pol delta) is a heterotrimeric enzyme consisting of Pol3 (the catalytic subunit), Pol31 and Pol32. New pol31 alleles were constructed by introducing mutations into conserved amino acid residues in all 10 identified regions of Pol31. Six novel temperature-sensitive (ts) or cold-sensitive (cs) alleles, carrying mutations in regions III, IV, VII, VIII or IX, conferred a range of defects in the response to replication stress or DNA damage. Deletion of SGS1, RAD52, SRS2, MRC1 or RAD24 had a deleterious effect only in combination with those pol31 alleles that had a phenotype as single mutants, suggesting a requirement for recombination and checkpoint functions in processing the DNA lesions or structures that form as a consequence of replication with a defective Pol delta. In contrast, deletion of POL32 negatively affected the growth of almost all pol31 mutants, suggesting an important role for all conserved amino acids of Pol31 in maintaining the integrity of Pol delta complex structurally, at least in the absence of the third subunit. Surprisingly, deletions of RAD18 and MGS1 aggravated the temperature sensitivity conferred by most ts or cs alleles and specifically suppressed the hys2-1 and hys2-1-like mutations of POL31. Deletion of RAD5 or MMS2 had an effect on pol31 ts/cs mutants similar to that of RAD18, whereas deletion of RAD30 or REV3 had no effect. We propose that Rad18/Rad5/Mms2 and Mgs1 are required to promote replication when forks are destabilized or stalled due to defects in Pol delta. These data are consistent with the biochemical activity of the human Mgs1 orthologue, which binds and stimulates Pol deltain vitro. We also demonstrate that Mgs1 interacts physically with Pol31 in vivo. Moreover, regions I and VII of Pol31, which are specifically sensitive to high levels of Mgs1 and PCNA, could be sites of interaction.

Adenosine Triphosphatases↗

PubMed2003

Elg1 forms an alternative PCNA-interacting RFC complex required to maintain genome stability.

BACKGROUND: Genome instability is a hallmark of cancer and plays a critical role in generating the myriad of phenotypes selected for during tumor progression. However, the mechanisms that prevent genome rearrangements remain poorly understood. RESULTS: To elucidate the mechanisms that ensure genome stability, we screened a collection of candidate genes for suppressors of gross chromosomal rearrangements (GCRs) in budding yeast. One potent suppressor gene encodes Elg1, a conserved but uncharacterized homolog of the large RFC subunit Rfc1 and the alternative RFC subunits Ctf18/Chl12 and Rad24. Our results are consistent with the hypothesis that Elg1 forms a novel and distinct RFC-like complex in both yeast and human cells. We find that Elg1 is required for efficient S phase progression and telomere homeostasis in yeast. Elg1 interacts physically with the PCNA homolog Pol30 and the FEN-1 homolog Rad27. The physical and genetic interactions suggest a role for Elg1 in Okazaki fragment maturation. Furthermore, Elg1 acts in concert with the alternative Rfc1-like proteins Rad24 and Ctf18 to enable Rad53 checkpoint kinase activation in response to replication stress. CONCLUSIONS: Collectively, these results reveal that Elg1 forms a novel and conserved alternative RFC complex. Furthermore, we propose that genome instability arises at high frequency in elg1 mutants due to a defect in Okazaki fragment maturation.

Amino Acid Sequence↗

PubMed2026

PARG inhibition reduces ssDNA levels and limits RPA loading upon replication fork collapse.

Poly(ADP-ribosyl)ation (PARylation) is a transient post-translational modification catalyzed by PARP enzymes and reversed by PARG. PARG inhibition causes sustained PARylation and is being explored as an anticancer strategy, but its cellular consequences remain incompletely understood. Here, we examine how persistent PARylation influences cellular responses to replication stress and DNA damage. We show that sustained PARylation reduces phosphorylated and chromatin-bound RPA most strongly under fork-stalling conditions that progress toward fork collapse. This effect requires PARP1 activity and is restrained by intact ATR-CHK1 signaling, as checkpoint inhibition renders otherwise resistant cells permissive for PARG inhibitor-associated phosphorylated RPA loss from the chromatin. The reduction of RPA phosphorylation is not dependent on BRCA1 and it is not accompanied by increased RAD51 loading. Instead, reduced chromatin-bound RPA coincides with decreased exposed ssDNA. Our results identify a checkpoint-dependent fork-collapse state in which sustained PARylation limits ssDNA and RPA levels.

Replication Protein A↗

PubMed2006

Genome-wide replication profiles of S-phase checkpoint mutants reveal fragile sites in yeast.

The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

Cell Cycle Proteins↗

PubMed2005

YB-1 represses AP1-dependent gene transactivation and interacts with an AP-1 DNA sequence.

Involvement of the AP-1 (activator protein-1) transcription factor has been demonstrated previously in the regulation of cell proliferation and cell-cycle progression, in the control of cell migration, invasion and metastasis, and in signal transduction, stress responsiveness, DNA replication and DNA repair. YB-1 (Y-box-binding protein-1) has also been implicated in many of these processes. However, the mechanism by which YB-1 mediates these processes is poorly understood. In the present study, we report that overexpression of a transfected gene encoding YB-1 in human HeLa cervical carcinoma cells significantly represses the transactivation of a minimal AP-1 reporter construct in response to the tumour promoter PMA. YB-1 also represses mRNA expression and PMA-induced promoter transactivation of the endogenous AP-1 target gene encoding matrix metalloproteinase-12 (metalloelastase). YB-1 transrepression of both the minimal and matrix metalloproteinase-12 promoter reporter constructs is dependent on the AP-1 sequence. To identify new nuclear proteins that bind specifically to the AP-1 DNA-binding site, we devised a DNA-affinity-chromatography-based assay termed NAPSTER (nucleotide-affinity preincubation specificity test of recognition) and discovered a 49 kDa protein from human cancer cells that binds in a sequence-specific manner to the AP-1 DNA sequence. By tandem MS fragmentation sequencing analyses we determined that p49 is a YB-1. Immunoblotting of the NAPSTER-purified p49 protein using anti-YB-1 antibodies confirmed YB-1 binding to the AP-1 DNA sequence, as did gel mobility-supershift assays using YB-1 antibodies. This is the first report of YB-1 transrepression and interaction at the AP-1 DNA-binding site.

Base Sequence↗

PubMed2004

A novel protein activity mediates DNA binding of an ATR-ATRIP complex.

The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.

Adaptor Proteins, Signal Transducing↗

PubMed2004

BRCA1 : BARD1 induces the formation of conjugated ubiquitin structures, dependent on K6 of ubiquitin, in cells during DNA replication and repair.

The N-terminus of the BRCA1 protein bears a RING finger domain that functions as an E3 ubiquitin ligase in vitro where it is able to catalyse the synthesis of monoubiquitin and polyubiquitin targeted proteins. This activity is greatly increased when BRCA1 is in a complex with its N-terminal binding partner BARD1. In this report we use an immunohistochemical approach to demonstrate the association of cellular BRCA1 with the end product of the ubiquitin conjugation and ligation pathway, conjugated ubiquitin. Association is apparent at DNA replication structures in S-phase and following treatment with hydroxyurea and also at sites of double strand break repair after exposure to ionizing radiation. Down-regulation of endogenous, cellular BRCA1 : BARD1 using siRNA results in abrogation of ubiquitin conjugation in these structures, suggesting that heterodimer activity is required for their formation. Conversely, ectopically expressed full-length BRCA1, but not BRCA1 bearing specific N-terminal amino acid substitutions, is able to cooperate with BARD1 to increase ubiquitin conjugation in cells. Conjugation of ubiquitin in foci is inhibited by the expression of ubiquitin bearing a lysine 6 mutation suggesting that the ubiquitin polymers formed at these sites are dependent on lysine-6 for linkage. Together these data demonstrate that BRCA1 directed ligation of ubiquitin occurs during S-phase and in response to replication stress and DNA damage and is therefore likely to be a significant aspect of BRCA1 cellular activity.

BRCA1 Protein↗

PubMed2003

Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Induction of the tumor-suppressor p16(INK4a) within regenerative epithelial crypts in ulcerative colitis.

p16(INK4a) is a major tumor-suppressor protein, but its regulation and settings of fuction remain poorly understood. To explore the notion that p16 is induced in vivo in response to replicative stress, we examined p16 expression in tissues from human ulcerative colitis (UC; n = 25) and normal controls (n = 20). p16 was expressed strongly in UC-associated neoplasms (n = 17), as seen previously in sporadic colonic neoplasms. In non-neoplastic UC epithelium, p16 was expressed in 33% of crypts (the proliferative compartment) compared to < 1% of normal controls. p16 expression did not correlate with degree of inflammation but did correlate with the degree of crypt architecture distortion (P = .002)-a reflection of epithelial regeneration. In coimmunofluorescence studies with Ki67, p16 expression was associated with cell cycle arrest (P < .001). Both UC and normal crypts displayed evidence for the activation of the DNA damage checkpoint pathway, and p16 was induced in primary cultures of normal epithelial cells by ionizing irradiation (IR). However, induction by IR displayed delayed kinetics, implying that p16 is not an immediate target of the checkpoint pathway. These findings support a model in which p16 is induced as an "emergency brake" in cells experiencing sustained replicative stress.

Colitis, Ulcerative↗

PubMed2005

DNA helicases as targets for anti-cancer drugs.

DNA helicases have essential roles in nucleic acid metabolism by facilitating cellular processes including replication, recombination, DNA repair, and transcription. The vital roles of helicases in these pathways are reflected by their emerging importance in the maintenance of genomic stability. Recently, a number of human diseases with cancer predisposition have been shown to be genetically linked to a specific helicase defect. This has led researchers to further investigate the roles of helicases in cancer biology, and to study the efficacy of targeting human DNA helicases for anti-cancer drug treatment. Helicase-specific inhibition in malignant cells may compromise the high proliferation rates of cancerous tissues. The role of RecQ helicases in response to replicational stress suggests a molecular target for selectively eliminating malignant tumor cells by a cancer chemotherapeutic agent. Alternate DNA secondary structures such as G-quadruplexes that may form in regulatory regions of oncogenes or G-rich telomere sequences are potential targets for cancer therapy since these sequence-specific structures are proposed to affect gene expression and telomerase activation, respectively. Small molecule inhibitors of G-quadruplex helicases may be used to regulate cell cycle progression by modulating promotor activation or disrupting telomere maintenance, important processes of cellular transformation. The design of small molecules which deter helicase function at telomeres may provide a molecular target since telomerase activity is necessary for the proliferation of numerous immortal cells. Although evidence suggests that helicases are specifically inhibited by certain DNA binding compounds, another area of promise in anti-cancer therapy is siRNA technology. Specific knockdown of helicase expression can be utilized as a means to sensitize oncogenic proliferating cell lines. This review will address these topics in detail and summarize the current avenues of research in anti-cancer therapy targeting helicases through small molecule inhibitors of DNA-protein complexes, DNA binding drugs, or down-regulation of helicase gene expression.

Animals↗

PubMed2006

Cdc7 is an active kinase in human cancer cells undergoing replication stress.

Cdc7 kinase promotes and regulates DNA replication in eukaryotic organisms. Multiple mechanisms modulating kinase activity in response to DNA replication stress have been reported, supporting the opposing notions that Cdc7 either plays an active role under these conditions or, conversely, is a final target inactivated by a checkpoint response. We have developed new immnunological reagents to study the properties of human Cdc7 kinase in cells challenged with the ribonucleotide reductase inhibitor hydroxyurea or with the DNA topoisomerase II inhibitor etoposide. We show that Cdc7.Dbf4 and Cdc7.Drf1 complexes are stable and active in multiple cell lines upon drug treatment, with Cdc7.Dbf4 accumulating on chromatin-enriched fractions. Cdc7 depletion by small interfering RNA in hydroxyurea and etoposide impairs hyper-phosphorylation of Mcm2 at specific Cdc7-dependent phosphorylation sites and drug-induced hyper-phosphorylation of chromatin-bound Mcm4. Furthermore, sustained inhibition of Cdc7 in the presence of these drugs increases cell death supporting the notion that the Cdc7 kinase plays a role in maintaining cell viability during replication stress.

Apoptosis↗

PubMed1985

Outcomes of role stress: a multisample constructive replication.

Responses from four separate samples of accountants and hospital employees provided a constructive replication of the Bedeian and Armenakis (1981) model of the causal nexus between role stress and selected outcome variables. We investigated the relationship between both role ambiguity and role conflict--as specific forms of role stress--and job-related tension, job satisfaction, and propensity to leave, using LISREL IV, a technique capable of providing statistical data for a hypothesized population model, as well as for specific causal paths. Results, which support the Bedeian and Armenakis model, are discussed in light of previous research.

Analysis of Variance↗

PubMed2001

Mrc1 transduces signals of DNA replication stress to activate Rad53.

Cells experiencing DNA replication stress activate a response pathway that delays entry into mitosis and promotes DNA repair and completion of DNA replication. The protein kinases ScRad53 and SpCds1 (in baker's and fission yeast, respectively) are central to this pathway. We describe a conserved protein Mrc1, mediator of the replication checkpoint, required for activation of ScRad53 and SpCds1 during replication stress. mrc1 mutants are sensitive to hydroxyurea and have a checkpoint defect similar to rad53 and cds1 mutants. Mrc1 may be the replicative counterpart of Rad9 and Crb2, which are required for activating ScRad53 and Chk1 in response to DNA damage.

Amino Acid Sequence↗

PubMed2005

DNA replication stress-induced phosphorylation of cyclic AMP response element-binding protein mediated by ATM.

The DNA damage-response regulators ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) are structurally and functionally related protein kinases that exhibit nearly identical substrate specificities in vitro. Current paradigms hold that the relative contributions of ATM and ATR to nuclear substrate phosphorylation are dictated by the type of initiating DNA lesion; ATM-dependent substrate phosphorylation is principally activated by DNA double strand breaks, whereas ATR-dependent substrate phosphorylation is induced by UV light and other forms of DNA replication stress. In this report, we employed the cyclic AMP-response element-binding (CREB) protein to provide evidence for substrate discrimination by ATM and ATR in cellulo. ATM and ATR phosphorylate CREB in vitro, and CREB is phosphorylated on Ser-121 in intact cells in response to ionizing radiation (IR), UV light, and hydroxyurea. The UV light- and hydroxyurea-induced phosphorylation of CREB was delayed in comparison to the canonical ATR substrate CHK1, suggesting potentially different mechanisms of phosphorylation. UV light-induced CREB phosphorylation temporally correlated with ATM autophosphorylation on Ser-1981, and an ATM-specific small interfering RNA suppressed CREB phosphorylation in response to this stimulus. UV light-induced CREB phosphorylation was absent in ATM-deficient cells, confirming that ATM is required for CREB phosphorylation in UV irradiation-damaged cells. Interestingly, RNA interference-mediated suppression of ATR partially inhibited CREB phosphorylation in response to UV light, which correlated with reduced phosphorylation of ATM on Ser-1981. These findings suggest that ATM is the major genotoxin-induced CREB kinase in mammalian cells and that ATR lies upstream of ATM in a UV light-induced signaling pathway.

Ataxia Telangiectasia↗

PubMed2003

DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells.

Expression of adenovirus E1A deregulates cell proliferation to facilitate viral DNA replication, prompting the initiation of apoptosis signaled primarily through proapoptotic BAK in productively infected cells. We demonstrate here that in uninfected cells, BAK is complexed with the anti-apoptotic BCL-2 family member Myeloid Cell Leukemia 1 (MCL-1). E1A expression during infection resulted in the specific down-regulation of MCL-1 through destabilization of the protein and loss of the mRNA. Upon loss of the MCL-1-BAK complex, BAK complexed with either BAX in proapoptotic E1B mutant adenovirus-infected cells, or with the adenovirus BCL-2 homolog E1B 19K in cells infected with the wild-type virus in which apoptosis is inhibited. Loss of MCL-1 was required to initiate the apoptotic pathway in infected cells as restoration of MCL-1 expression rescued infected cells from E1A-induced apoptosis. Analogous to E1A expression, DNA damage down-regulates MCL-1, and adenovirus infection resulted in the accumulation of phosphorylated H2AX and ataxia-telangiectasia mutant protein (ATM), hallmarks of DNA double-strand breaks. Thus, MCL-1 may function by maintaining BAK in an inactive state, and the loss of MCL-1 upon activation of the DNA damage response, perhaps through replication stress induced in virus infected cells, may be required to initiate the apoptotic response.

Adenoviridae↗

PubMed2026

RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability.

The nucleolus is organized around actively transcribed ribosomal RNA genes (rDNA), where high RNA polymerase I (Pol I) activity creates intrinsic susceptibility to replication stress and DNA damage. Here, we identify the DNA translocase RAD54L as a critical regulator of the nucleolar DNA damage response (nDDR) to rDNA double-strand breaks (DSBs) and replication stress. We show that RAD54L localizes to the nucleolus under basal conditions and is recruited to nucleolar caps following CRISPR-Cas9-induced rDNA-DSBs to promote repair. RAD54L loss results in persistent RAD51 foci, increased nucleolar γH2AX, and micronuclei formation, indicating defective resolution of rDNA lesions and genome instability. Under baseline conditions and replication stress induced by the Pol I transcription inhibitor CX-5461, RAD54L limits the accumulation of ssDNA and coordinates nDDR signaling. We further show that rDNA-DSBs induce RNA polymerase II-dependent RNA-DNA hybrids (R-loops) at intergenic rDNA regions, which facilitate nucleolar reorganization and cap formation and repair factor recruitment. Together, these findings establish RAD54L as a key regulator that coordinates replication stress response and rDNA repair, maintaining rDNA stability and genome integrity.

DNA, Ribosomal↗

PubMed2006

Polo-like kinase-1 controls proteasome-dependent degradation of Claspin during checkpoint recovery.

DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint kinase ATR phosphorylates and activates its effector kinase Chk1 in a process that critically depends on Claspin . However, it is not known how exactly this kinase cascade is silenced. Here we demonstrate that the abundance of Claspin is regulated through proteasomal degradation. In response to DNA damage, Claspin is transiently stabilized, and its expression depends on Chk1 kinase activity. In addition, we show that Claspin is degraded upon mitotic entry, a process that depends on the beta-TrCP-SCF ubiquitin ligase and Polo-like kinase-1 (Plk1). We demonstrate that Claspin interacts with both beta-TrCP and Plk1 and that inactivation of these components or the beta-TrCP recognition motif in Claspin prevents its mitotic degradation. Interestingly, expression of a nondegradable Claspin mutant inhibits recovery from a DNA-damage-induced checkpoint arrest. Thus, we conclude that Claspin levels are tightly regulated, both during unperturbed cell cycles and after DNA damage. Moreover, our data demonstrate that the degradation of Claspin at the onset of mitosis is an essential step for the recovery of a cell from a DNA-damage-induced cell-cycle arrest.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Mammalian TIMELESS and Tipin are evolutionarily conserved replication fork-associated factors.

The function of the mammalian TIMELESS protein (TIM) has been enigmatic. TIM is essential for early embryonic development, but little is known regarding its biochemical and cellular function. Although identified based on similarity to a Drosophila circadian clock factor, it also shares similarity with a second family of proteins that is more widely conserved throughout eukaryotes. Members of this second protein family in yeast (S.c. Tof1p, S.p. Swi1p) have been implicated in DNA synthesis, S-phase-dependent checkpoint activation and chromosome cohesion, three processes coordinated at the level of the replication fork complex. The present work demonstrates that mammalian TIM and its constitutive binding partner, Tipin (ortholog of S.c. Csm3p, S.p. Swi3p), are replisome-associated proteins. Both proteins associate with components of the endogenous replication fork complex, and are present at BrdU-positive DNA replication sites. Knock-down of TIM also compromises DNA replication efficiency. Further, the direct binding of the TIM-Tipin complex to the 34 kDa subunit of replication protein A provides a biochemical explanation for the potential coupling role of these proteins. Like TIM, Tipin is also involved in the molecular mechanism of UV-dependent checkpoint activation and cell growth arrest. Tipin additionally associates with peroxiredoxin2 and appears to be involved in checkpoint responses to H(2)O(2), a role recently described for yeast versions of TIM and Tipin. Together, this work establishes TIM and Tipin as functional orthologs of their replisome-associated yeast counterparts capable of coordinating replication with genotoxic stress responses, and distinguishes mammalian TIM from the circadian-specific paralogs from which it was originally identified.

Animals↗