RESUMO
Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/genética , Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Quinase 1 do Ponto de Checagem , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA , Dimerização , Células HeLa , Humanos , Modelos Biológicos , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Frações Subcelulares/metabolismoRESUMO
UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fase S/efeitos da radiação , Raios Ultravioleta , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem , DNA/metabolismo , Proteínas de Ligação a DNA , Regulação para Baixo/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Células HeLa , Humanos , Idoxuridina , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Ligação Proteica/efeitos da radiação , Proteínas Quinases/metabolismo , Proteína de Replicação A/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of skin cancers. Seven complementation groups, corresponding to seven proteins involved in nucleotide excision repair (NER), are associated with this syndrome. However, in XP variant patients, the disorder is caused by defects in DNA polymerase eta; this error prone polymerase, encoded by POLH, is involved in translesion DNA synthesis (TLS) on DNA templates damaged by ultraviolet light (UV). We constructed a recombinant adenovirus carrying the human POLH cDNA linked to the EGFP reporter gene (AdXPV-EGFP) and infected skin fibroblasts from both XPV and XPA patients. Twenty-four hours after infection, the DNA polymerase eta-EGFP fusion protein was detected by Western blot analysis, demonstrating successful transduction by the adenoviral vector. Protein expression was accompanied by reduction in the high sensitivity of XPV cells to UV, as determined by cell survival and apoptosis-induction assays. Moreover, the pronounced UV-induced inhibition of DNA synthesis in XPV cells and their arrest in S phase were attenuated in AdXPV-EGFP infected cells, confirming that the transduced polymerase was functional. However, over-expression of polymerase eta mediated by AdXPV-EGFP infection did not result in enhancement of cell survival, prevention of apoptosis, or higher rate of nascent DNA strand growth in irradiated XPA cells. These results suggest that TLS by DNA polymerase eta is not a limiting factor for recovery from cellular responses induced by UV in excision-repair deficient fibroblasts.
Assuntos
Adenoviridae/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Vetores Genéticos/genética , Transdução Genética/métodos , Xeroderma Pigmentoso/genética , Western Blotting , Células Cultivadas , Replicação do DNA/efeitos da radiação , DNA Complementar/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Raios UltravioletaRESUMO
Inhibition of replicon initiation is a stereotypic DNA damage response mediated through S checkpoint mechanisms not yet fully understood. Studies were undertaken to elucidate the function of checkpoint proteins in the inhibition of replicon initiation following irradiation with 254 nm UV light (UVC) of diploid human fibroblasts immortalized by the ectopic expression of telomerase. Velocity sedimentation analysis of nascent DNA molecules revealed a 50% inhibition of replicon initiation when normal human fibroblasts were treated with a low dose of UVC (1 J/m(2)). Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder fibroblasts, which lack an S checkpoint response when exposed to ionizing radiation, responded normally when exposed to UVC and inhibited replicon initiation. Pretreatment of normal and AT fibroblasts with caffeine or UCN-01, inhibitors of ATR (AT mutated and Rad3 related) and Chk1, respectively, abolished the S checkpoint response to UVC. Moreover, overexpression of kinase-inactive ATR in U2OS cells severely attenuated UVC-induced Chk1 phosphorylation and reversed the UVC-induced inhibition of replicon initiation, as did overexpression of kinase-inactive Chk1. Taken together, these data suggest that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR signaling through Chk-1 and is independent of ATM, Nbs1, and Mre11.
Assuntos
Replicação do DNA , DNA/efeitos da radiação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Replicon/genética , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem , DNA/genética , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA , Doxiciclina/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Ativação Enzimática , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Fibroblastos/fisiologia , Fibroblastos/efeitos da radiação , Genes cdc , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Telomerase/metabolismo , Proteínas Supressoras de Tumor , Raios UltravioletaRESUMO
Although BRAF V600 mutations in melanocytic tumors are not UV-signature mutations, it is plausible that they could still arise from error-prone replication of UV-damaged DNA. We propose a mechanism for their origin, taking into consideration melanocytic-specific BRAF tandem mutations, nearby potential pyrimidine dimer sites, the properties of specialized DNA polymerases, and biological selection.
Assuntos
Dano ao DNA , Melanoma/genética , Neoplasias Induzidas por Radiação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Humanos , Melanoma/epidemiologia , Neoplasias Induzidas por Radiação/epidemiologia , Fatores de Risco , Neoplasias Cutâneas/epidemiologia , Raios UltravioletaRESUMO
Telomerase-immortalized lines of diploid xeroderma pigmentosum variant (XP-V) fibroblasts (XP115LO and XP4BE) were complemented for constitutive or regulated expression of wild-type human DNA polymerase eta (hpol eta). The ectopic gene was expressed from a retroviral LTR at a population average of 34- to 59-fold above the endogenous (mutated) mRNA and high levels of hpol eta were detected by immunoblotting. The POLH cDNA was also cloned downstream from an ecdysone-regulated promoter and transduced into the same recipient cells. Abundance of the wild-type mRNA increased approximately 10-fold by addition of ponasterone to the culture medium. Complemented cell lines acquired normal resistance to the cytotoxic effects of UVC, even in the presence of 1mM caffeine. They also tolerated higher levels of UVC-induced template lesions during nascent DNA elongation when compared to normal fibroblasts (NHF). UVC-induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus were measured in the XP115LO+XPV cell line overproducing hpol eta constitutively (E. Bassett, N.M. King, M.F. Bryant, S. Hector, L. Pendyala, S.G. Chaney, M. Cordeiro-Stone, The role of DNA polymerase eta in translesion synthesis past platinum-DNA adducts in human fibroblasts, Cancer Res. 64 (2004) 6469-6475). Induced mutation frequencies were significantly reduced, even below those observed in NHF; however, the average mutation frequency in untreated cultures was about three-fold higher than in the isogenic vector-control cell line. In this study, spontaneous HPRT mutation frequencies were measured at regular intervals, as isogenic fibroblasts either lacking or overproducing hpol eta were expanded for 100 population doublings. The mutation rates estimated from these results were not significantly increased in XP115LO cells expressing abnormal levels of hpol eta, relative to the cells lacking this specialized polymerase. These findings suggest that diploid human fibroblasts with normal DNA repair capacities and intact checkpoints are well protected against the potential mutagenic outcome of overproducing hpol eta, while still benefiting from accurate translesion synthesis of UV-induced pyrimidine dimers.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Diploide , Fibroblastos/enzimologia , Mutação da Fase de Leitura , Western Blotting , Cafeína/farmacologia , Linhagem Celular Transformada , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Dosagem de Genes , Teste de Complementação Genética , Variação Genética , Humanos , Hipoxantina Fosforribosiltransferase/genética , Cinética , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Raios Ultravioleta , Xeroderma Pigmentoso/enzimologia , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologiaRESUMO
Cisplatin, a widely used chemotherapeutic agent, has been implicated in the induction of secondary tumors in cancer patients. This drug is presumed to be mutagenic because of error-prone translesion synthesis of cisplatin adducts in DNA. Oxaliplatin is effective in cisplatin-resistant tumors, but its mutagenicity in humans has not been reported. The polymerases involved in bypass of cisplatin and oxaliplatin adducts in vivo are not known. DNA polymerase eta is the most efficient polymerase for bypassing platinum adducts in vitro. We evaluated the role of polymerase eta in translesion synthesis past platinum adducts by determining cytotoxicity and induced mutation frequencies at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in diploid human fibroblasts. Normal human fibroblasts (NHF1) were compared with xeroderma pigmentosum variant (XPV) cells (polymerase eta-null) after treatment with cisplatin. In addition, XPV cells complemented for polymerase eta expression were compared with the isogenic cells carrying the empty expression vector. Cytotoxicity and induced mutagenicity experiments were measured in parallel in UVC-irradiated fibroblasts. We found that equitoxic doses of cisplatin induced mutations in fibroblasts lacking polymerase eta at frequencies 2- to 2.5-fold higher than in fibroblasts with either normal or high levels of polymerase eta. These results indicate that polymerase eta is involved in error-free translesion synthesis past some cisplatin adducts. We also found that per lethal event, cisplatin was less mutagenic than UVC. Treatment with a wide range of cytotoxic doses of oxaliplatin did not induce mutations above background levels in cells either expressing or lacking polymerase eta, suggesting that oxaliplatin is nonmutagenic in human fibroblasts.
Assuntos
Cisplatino/farmacologia , Adutos de DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Fibroblastos/enzimologia , Compostos Organoplatínicos/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Cisplatino/metabolismo , DNA/genética , DNA Polimerase Dirigida por DNA/biossíntese , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Mutação da Fase de Leitura , Deleção de Genes , Humanos , Hipoxantina Fosforribosiltransferase/genética , Masculino , Compostos Organoplatínicos/metabolismo , Oxaliplatina , Raios Ultravioleta , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologiaRESUMO
The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.
Assuntos
Melanócitos/citologia , Melanócitos/efeitos da radiação , Melanoma/patologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos da radiação , Raios Ultravioleta , Biomarcadores/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem , Dano ao DNA , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/metabolismo , Diploide , Relação Dose-Resposta à Radiação , Fibroblastos/efeitos da radiação , Humanos , Melaninas/metabolismo , Fosforilação/efeitos da radiação , Proteínas Quinases/metabolismo , Dímeros de Pirimidina/metabolismoRESUMO
We investigated the hypothesis that the strength of the activation of the intra-S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m(-2) UVC in early, mid- and late-S phase. The endpoints measured were the following: (1) radio-resistant DNA synthesis (RDS), (2) induction of Chk1 phosphorylation, (3) initiation of new replicons and (4) length of replication tracks synthesized after irradiation. RDS analysis showed that global DNA synthesis was inhibited by approximately the same extent (30 ± 12%), regardless of when during S phase the fibroblasts were exposed to UVC. Western blot analysis revealed that the UVC-induced phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 was high in early and mid S but 10-fold lower in late S. DNA fiber immunostaining studies indicated that the replication fork displacement rate decreased in irradiated cells at the three time points examined; however, replicon initiation was inhibited strongly in early and mid S, but this response was attenuated in late S. These results suggest that the intra-S checkpoint activated by UVC-induced DNA damage is not as robust toward the end of S phase in its inhibition of the latest firing origins in human fibroblasts.
Assuntos
Dano ao DNA , Diploide , Fase S , Replicação do DNA , Fibroblastos/citologia , HumanosRESUMO
In vitro replication assays for detection and quantification of bypass of UV-induced DNA photoproducts were used to compare the capacity of extracts prepared from different human cell lines to replicate past the cis,syn cyclobutane thymine dimer ([c,s]TT). The results demonstrated that neither nucleotide excision repair (NER) nor mismatch repair (MMR) activities in the intact cells interfered with measurements of bypass replication efficiencies in vitro. Extracts prepared from HeLa (NER- and MMR-proficient), xeroderma pigmentosum group A (NER-deficient), and HCT116 (MMR-deficient) cells displayed similar capacity for translesion synthesis, when the substrate carried the site-specific [c,s]TT on the template for the leading or the lagging strand of nascent DNA. Extracts from xeroderma pigmentosum variant cells, which lack DNA polymerase eta, were devoid of bypass activity. Bypass-proficient extracts as a group (n=16 for 3 extracts) displayed higher efficiency (P=0.005) for replication past the [c,s]TT during leading strand synthesis (84+/-22%) than during lagging strand synthesis (64+/-13%). These findings are compared to previous results concerning the bypass of the (6-4) photoproduct [Biochemistry 40 (2001) 15215] and analyzed in the context of the reported characteristics of bypass DNA polymerases implicated in translesion synthesis of UV-induced DNA lesions. Models to explain how these enzymes might interact with the DNA replication machinery are considered. An alternative pathway of bypass replication, which avoids translesion synthesis, and the mutagenic potential of post-replication repair mechanisms that contribute to the duplication of the human genome damaged by UV are discussed.
Assuntos
Dano ao DNA , Replicação do DNA , Dímeros de Pirimidina/metabolismo , Dímeros de Pirimidina/efeitos da radiação , Linhagem Celular , Reparo do DNA , Células HeLa , Humanos , Modelos Biológicos , Mutagênese , Raios Ultravioleta/efeitos adversos , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismoRESUMO
The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21(Cip1/Waf1) post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase eta, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol eta protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.
Assuntos
Cafeína/farmacologia , Ciclo Celular/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , DNA/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular , Linhagem Celular , Quinase 1 do Ponto de Checagem , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , DNA/genética , DNA/efeitos da radiação , Dano ao DNA , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA , DNA Polimerase Dirigida por DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Zíper de Leucina , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor , Raios UltravioletaRESUMO
This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra-S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6-4 pyrimidine-pyrimidone photoproducts was highest in DNA from UVC-irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA-UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8-oxo-7,8-dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR.
Assuntos
Biomarcadores/análise , Fibroblastos/efeitos da radiação , Dímeros de Pirimidina/análise , Raios Ultravioleta , Humanos , Immunoblotting , Dímeros de Pirimidina/efeitos da radiação , Efeitos da RadiaçãoRESUMO
The ATR/CHK1-dependent intra-S checkpoint inhibits replicon initiation and replication fork progression in response to DNA damage caused by UV (UV) radiation. It has been proposed that this signaling cascade protects against UV-induced mutations by reducing the probability that damaged DNA will be replicated before it can be repaired. Normal human fibroblasts (NHF) were depleted of ATR or CHK1, or treated with the CHK1 kinase inhibitor TCS2312, and the UV-induced mutation frequency at the HPRT locus was measured. Despite clear evidence of S-phase checkpoint abrogation, neither ATR/CHK1 depletion nor CHK1 inhibition caused an increase in the UV-induced HPRT mutation frequency. These results question the premise that the UV-induced intra-S checkpoint plays a prominent role in protecting against UV-induced mutagenesis.
Assuntos
Fibroblastos/metabolismo , Mutagênese/efeitos da radiação , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Raios Ultravioleta/efeitos adversos , Linhagem Celular , Quinase 1 do Ponto de Checagem , Dano ao DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Fibroblastos/citologia , Loci Gênicos , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Mutação , Proteínas Quinases/genética , Proteínas Quinases/metabolismoRESUMO
The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which are mediated by the TIMELESS-TIPIN complex and CLASPIN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS-TIPIN complex and CLASPIN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS, TIPIN or CLASPIN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASPIN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS-TIPIN and CLASPIN have functions for preservation of intrinsic chromosomal stability that is separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Aberrações Cromossômicas , Replicação do DNA/fisiologia , Instabilidade Genômica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/deficiência , Quinase 1 do Ponto de Checagem , Proteínas de Ligação a DNA , Fibroblastos , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Funções Verossimilhança , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Proteínas Quinases/deficiência , Proteínas Serina-Treonina Quinases/deficiênciaAssuntos
Transformação Celular Neoplásica/genética , Replicação do DNA/fisiologia , Neoplasias/genética , Fase S/genética , Animais , Carcinógenos , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/patologia , Humanos , Neoplasias/induzido quimicamente , Neoplasias/etiologia , Neoplasias/patologiaRESUMO
The in vitro replication assay described here measures bidirectional replication of a circular double- stranded DNA template upon initiation at the SV40 origin. It models a single eukaryotic replication unit (replicon) and recapitulates the biochemical steps involved in the catalysis of both leading and lagging strand synthesis during semiconservative DNA replication. Except for the SV40 large T antigen, all other proteins necessary for initiation and assembly of functional replication forks are provided by the cell-free extract. This assay can be used to demonstrate bypass replication of genotoxic lesions. It supports replication across a specific damaged site on the template DNA (i.e., translesion synthesis) by specialized DNA polymerases. This chapter illustrates the efficient translesion synthesis of UV-induced thymine dimers by DNA polymerase eta.
Assuntos
Dano ao DNA , Replicação do DNA , DNA/biossíntese , DNA/genética , Animais , Sequência de Bases , Sistema Livre de Células , DNA/isolamento & purificação , Eletroforese em Gel de Ágar , Células HeLa , HumanosRESUMO
Following acquisition of DNA damage S-phase progression may potentially be affected via multiple mechanisms. For example DNA damage-activated signal transduction pathways negatively regulate the initiation of DNA synthesis at unfired origins of replication, a process termed the 'S-phase checkpoint' or the 'intra-S-phase checkpoint'. Additionally, many DNA lesions pose physical barriers to replication forks and therefore inhibit DNA synthesis directly by blocking the elongation of active replicons. Inhibition of DNA synthesis in response to DNA damage is commonly assayed by measuring incorporation of radiolabeled or halogenated nucleotides into bulk genomic DNA. However, these techniques do not distinguish between effects of DNA damage on initiation and elongation phases of DNA synthesis. The velocity sedimentation protocol described here allows investigators to determine the effects of DNA damage on initiation and elongation events. This technique involves labeling replicating DNA with (3)H-thymidine, then analyzing the size distribution of labeled ssDNAs based on their differential density sedimentation profiles after centrifugation through alkaline sucrose gradients. Determining the relative abundance and growth rates of small nascent ssDNAs provides an index of initiation and elongation events, respectively. Therefore, analysis of replication dynamics using velocity sedimentation provides a potentially valuable tool for assaying S-phase checkpoints as well as other aspects of DNA replication.
Assuntos
Fracionamento Químico/métodos , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Mutagênicos/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase S do Ciclo Celular/genética , Linhagem Celular , Dano ao DNA/genética , Dano ao DNA/fisiologia , HumanosRESUMO
The Timeless-Tipin complex and Claspin are mediators of the ATR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. Tim-Tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process. Some models of the establishment of SCC posit that interactions between cohesin rings and replisomes could result in physiological replication stress requiring fork stabilization. The contributions of Timeless, Tipin, Claspin, Chk1 and ATR to SCC were investigated in genetically stable, human diploid fibroblast cell lines. Whereas Timeless, Tipin and Claspin showed similar contributions to UVC-induced activation of Chk1, siRNA-mediated knockdown of Timeless induced a 100-fold increase in sister chromatid discohesion, whereas the inductive effects of knocking down Tipin, Claspin and ATR were 4-20-fold. Knockdown of Chk1 did not significantly affect SCC. Consistent findings were obtained in two independently derived human diploid fibroblast lines and support a conclusion that SCC in human cells is strongly dependent on Timeless but independent of Chk1. Furthermore, the 10-fold difference in discohesion observed when depleting Timeless versus Tipin indicates that Timeless has a function in SCC that is independent of the Tim-Tipin complex, even though the abundance of Timeless is reduced when Tipin is targeted for depletion. A better understanding of how Timeless, Tipin and Claspin promote SCC will elucidate non-checkpoint functions of these proteins at DNA replication forks and inform models of the establishment of SCC.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem , Replicação do DNA , Proteínas de Ligação a DNA , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metáfase , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Raios Ultravioleta , CoesinasRESUMO
The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fase G2 , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quinase do Ponto de Checagem 2 , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dicetopiperazinas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Mitose , Fosforilação , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Inibidores da Topoisomerase II/farmacologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: The GINS complex is thought to be essential for the processes of initiation and elongation of DNA replication. This complex contains four subunits, one of which (Psf1) is proposed to bind to both chromatin and DNA replication-associated proteins. To date there have been no microscopic analyses to evaluate the chromatin distribution of this complex. Here, we show the organization of GINS complexes on extended chromatin fibers in relation to sites of DNA replication and replication-associated proteins. RESULTS: Using immunofluorescence microscopy we were able to visualize ORC1, ORC2, PCNA, and GINS complex proteins Psf1 and Psf2 bound to extended chromatin fibers. We were also able to detect these proteins concurrently with the visualization of tracks of recently replicated DNA where EdU, a thymidine analog, was incorporated. This allowed us to assess the chromatin association of proteins of interest in relation to the process of DNA replication. ORC and GINS proteins were found on chromatin fibers before replication could be detected. These proteins were also associated with newly replicated DNA in bead-like structures. Additionally, GINS proteins co-localized with PCNA at sites of active replication. CONCLUSION: In agreement with its proposed role in the initiation of DNA replication, GINS proteins associated with chromatin near sites of ORC binding that were devoid of EdU (absence of DNA replication). The association of GINS proteins with PCNA was consistent with a role in the process of elongation. Additionally, the large size of our chromatin fibers (up to approximately 7 Mb) allowed for a more expansive analysis of the distance between active replicons than previously reported.