RESUMO
Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη(-/-)/POLζ(-/-) cells from the chicken DT40 cell line. POLζ(-/-) cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Polη plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLη(-/-)/POLζ(-/-) cells shows that, unexpectedly, the loss of Polη significantly rescued all mutant phenotypes of POLζ(-/-) cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Polη contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells.
Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA/genética , Mutação , Animais , Antineoplásicos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Galinhas , Cisplatino/farmacologia , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Células HEK293 , Humanos , Metanossulfonato de Metila/farmacologia , Modelos Genéticos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Supressão Genética , Raios UltravioletaRESUMO
In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells.
Assuntos
Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Fase S , Alelos , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Galinhas , Inibidores de Cisteína Proteinase/farmacologia , Geminina , Células HeLa , Humanos , Leupeptinas/farmacologia , Modelos Biológicos , Mutação , Purinas/farmacologia , RoscovitinaRESUMO
In eukaryotic nuclei, DNA is wrapped around an octamer of core histones to form nucleosomes, and chromatin fibers are thought to be stabilized by linker histones of the H1 type. Higher eukaryotes express multiple variants of histone H1; chickens possess six H1 variants. Here, we generated and analyzed the phenotype of a complete deletion of histone H1 genes in chicken cells. The H1-null cells showed decreased global nucleosome spacing, expanded nuclear volumes, and increased chromosome aberration rates, although proper mitotic chromatin structure appeared to be maintained. Expression array analysis revealed that the transcription of multiple genes was affected and was mostly downregulated in histone H1-deficient cells. This report describes the first histone H1 complete knockout cells in vertebrates and suggests that linker histone H1, while not required for mitotic chromatin condensation, plays important roles in nucleosome spacing and interphase chromatin compaction and acts as a global transcription regulator.
Assuntos
Histonas/fisiologia , Nucleossomos/química , Animais , Ciclo Celular , Linhagem Celular , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Cromatina/ultraestrutura , Aberrações Cromossômicas , Histonas/genética , Interfase/genética , Mutação , Transcrição GênicaRESUMO
Homologous recombination (HR) is initiated by DNA double-strand breaks (DSB). However, it remains unclear whether single-strand lesions also initiate HR in genomic DNA. Chicken B lymphocytes diversify their Immunoglobulin (Ig) V genes through HR (Ig gene conversion) and non-templated hypermutation. Both types of Ig V diversification are initiated by AID-dependent abasic-site formation. Abasic sites stall replication, resulting in the formation of single-stranded gaps. These gaps can be filled by error-prone DNA polymerases, resulting in hypermutation. However, it is unclear whether these single-strand gaps can also initiate Ig gene conversion without being first converted to DSBs. The Mre11-Rad50-Nbs1 (MRN) complex, which produces 3' single-strand overhangs, promotes the initiation of DSB-induced HR in yeast. We show that a DT40 line expressing only a truncated form of Nbs1 (Nbs1(p70)) exhibits defective HR-dependent DSB repair, and a significant reduction in the rate--though not the fidelity--of Ig gene conversion. Interestingly, this defective gene conversion was restored to wild type levels by overproduction of Escherichia coli SbcB, a 3' to 5' single-strand-specific exonuclease, without affecting DSB repair. Conversely, overexpression of chicken Exo1 increased the efficiency of DSB-induced gene-targeting more than 10-fold, with no effect on Ig gene conversion. These results suggest that Ig gene conversion may be initiated by single-strand gaps rather than by DSBs, and, like SbcB, the MRN complex in DT40 may convert AID-induced lesions into single-strand gaps suitable for triggering HR. In summary, Ig gene conversion and hypermutation may share a common substrate-single-stranded gaps. Genetic analysis of the two types of Ig V diversification in DT40 provides a unique opportunity to gain insight into the molecular mechanisms underlying the filling of gaps that arise as a consequence of replication blocks at abasic sites, by HR and error-prone polymerases.
Assuntos
Linfócitos B/metabolismo , Quebras de DNA de Cadeia Simples , Região Variável de Imunoglobulina/genética , Proteínas Nucleares/metabolismo , Recombinação Genética , Animais , Linhagem Celular Tumoral , Galinhas , Reparo do DNA , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Conversão Gênica , Região Variável de Imunoglobulina/metabolismo , Proteínas Nucleares/genéticaRESUMO
Fbh1 (F-box DNA helicase 1) orthologues are conserved from Schizosaccharomyces pombe to chickens and humans. Here, we report the disruption of the FBH1 gene in DT40 cells. Although the yeast fbh1 mutant shows an increase in sensitivity to DNA damaging agents, FBH1(-)(/)(-) DT40 clones show no prominent sensitivity, suggesting that the loss of FBH1 might be compensated by other genes. However, FBH1(-)(/)(-) cells exhibit increases in both sister chromatid exchange and the formation of radial structures between homologous chromosomes without showing a defect in homologous recombination. This phenotype is reminiscent of BLM(-)(/)(-) cells and suggests that Fbh1 may be involved in preventing extensive strand exchange during homologous recombination. In addition, disruption of RAD54, a major homologous recombination factor in FBH1(-)(/)(-) cells, results in a marked increase in chromosome-type breaks (breaks on both sister chromatids at the same place) following replication fork arrest. Further, FBH1BLM cells showed additive increases in both sister chromatid exchange and the formation of radial chromosomes. These data suggest that Fbh1 acts in parallel with Bloom helicase to control recombination-mediated double-strand-break repair at replication blocks and to reduce the frequency of crossover.
Assuntos
Adenosina Trifosfatases/metabolismo , Troca Genética , DNA Helicases/metabolismo , Replicação do DNA , Vertebrados/metabolismo , Animais , Camptotecina/farmacologia , Galinhas , Cisplatino/farmacologia , Troca Genética/efeitos dos fármacos , Troca Genética/efeitos da radiação , Dano ao DNA , DNA Helicases/deficiência , DNA Helicases/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Raios gama , Deleção de Genes , Marcação de Genes , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Metanossulfonato de Metila/farmacologia , Modelos Genéticos , Dados de Sequência Molecular , Mutação/genética , RecQ Helicases , Raios UltravioletaRESUMO
REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.
Assuntos
Dano ao DNA , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Viral , Galinhas , Chlorocebus aethiops , DNA Polimerase Dirigida por DNA , Glutationa Transferase/metabolismo , Dados de Sequência Molecular , Nucleotidiltransferases/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Raios UltravioletaRESUMO
Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, gammaH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , Leupeptinas/farmacologia , Inibidores de Proteassoma , Recombinação Genética/efeitos dos fármacos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Genes BRCA1 , Células HeLa , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Rad51 Recombinase/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
In Saccharomyces cerevisiae, the linker histone HHO1 is involved in DNA repair. In higher eukaryotes, multiple variants of linker histone H1 exist but their involvement in the DNA damage response is unknown. To address this issue, we examined sensitivity to genotoxic agents in chicken DT40 cells lacking specific H1 variants. Among the six H1 variant mutants, only H1R(-/-) DT40 cells exhibited increased sensitivity to the alkylating agent methyl-methanesulfonate (MMS). The MMS sensitivity of H1R(-/-) cells was not enhanced by inactivation of Rad54. H1R(-/-) DT40 cells also exhibited: (i) a reduction in gene targeting efficiencies, (ii) impaired sister chromatid exchange, and (iii) an accumulation of IR-induced chromosomal aberrations at the G2 phase, all of which indicate the involvement of H1R in the Rad54-mediated homologous recombination (HR) pathway. The mobility of H1R but not H1L in the nucleus decreased after MMS treatment and the repair of double-stranded breaks generated by I-SceI was unaffected in H1R(-/-) cells, suggesting that H1R integrates into HR-mediated repair pathways at the chromosome structure level. Together, these findings provide the first genetic evidence that a specific H1 variant plays a unique and important role in the DNA damage response in vertebrates.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Variação Genética , Histonas/fisiologia , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Galinhas , Histonas/genética , Histonas/metabolismo , Metanossulfonato de Metila/farmacologia , Recombinação Genética , Troca de Cromátide Irmã , Fatores de TempoRESUMO
DNA polymerase lambda (Pol lambda) is a DNA polymerase beta (Pol beta)-like enzyme with both DNA synthetic and 5'-deoxyribose-5'-phosphate lyase domains. Recent biochemical studies implicated Pol lambda as a backup enzyme to Pol beta in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol lambda and Pol beta in BER of DNA damage in living cells, we disrupted the genes for both enzymes either singly or in combination in the chicken DT40 cell line and then characterized BER phenotypes. Disruption of the genes for both polymerases caused hypersensitivity to H(2)O(2)-induced cytotoxicity, whereas the effect of disruption of either polymerase alone was only modest. Similarly, BER capacity in cells after H(2)O(2) exposure was lower in Pol beta(-/-)/Pol lambda(-/-) cells than in Pol beta(-/-), wild-type, and Pol lambda(-/-) cells, which were equivalent. These results suggest that these polymerases can complement for one another in counteracting oxidative DNA damage. Similar results were obtained in assays for in vitro BER capacity using cell extracts. With MMS-induced cytotoxicity, there was no significant effect on either survival or BER capacity from Pol lambda gene disruption. A strong hypersensitivity and reduction in BER capacity was observed for Pol beta(-/-)/Pol lambda(-/-) and Pol beta(-/-) cells, suggesting that Pol beta had a dominant role in counteracting alkylation DNA damage in this cell system.
Assuntos
Dano ao DNA , DNA Polimerase beta/fisiologia , Reparo do DNA , Animais , Linhagem Celular , Sobrevivência Celular , Galinhas , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/farmacologia , Modelos Genéticos , NADP/metabolismo , Oxigênio/metabolismo , Plasmídeos/metabolismoRESUMO
One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.
Assuntos
Proteínas Aviárias/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Histonas/fisiologia , Rad51 Recombinase/metabolismo , Recombinação Genética , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Camptotecina/farmacologia , Células Cultivadas , Galinhas , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Raios gama , Instabilidade Genômica , Histonas/genética , Modelos Genéticos , Rad51 Recombinase/genética , TransfecçãoRESUMO
ASCIZ (ATMIN) was recently identified as a novel DNA damage response protein. Here we report that ASCIZ-deficient chicken DT40 B lymphocyte lines displayed markedly increased Ig gene conversion rates, whereas overexpression of human ASCIZ reduced Ig gene conversion below wild-type levels. However, neither the efficiency of double-strand break repair nor hypermutation was affected by ASCIZ levels, indicating that ASCIZ does not directly control homologous recombination or formation of abasic sites. Loss of ASCIZ led to mild sensitivity to the base damaging agent methylmethane sulfonate (MMS), yet remarkably, suppressed the dramatic MMS hypersensitivity of polbeta-deficient cells. These data suggest that ASCIZ may affect the choice between competing base repair pathways in a manner that reduces the amount of substrates available for Ig gene conversion.
Assuntos
Alquilantes/farmacologia , Proteínas de Transporte/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Conversão Gênica , Genes de Imunoglobulinas/genética , Animais , Proteínas de Transporte/genética , Linhagem Celular , DNA Polimerase beta/genética , Resistência a Medicamentos/genética , Humanos , Metanossulfonato de Metila/farmacologia , Camundongos , Camundongos Transgênicos , Mutagênicos/farmacologia , Proteínas Nucleares , Supressão Genética , Fatores de TranscriçãoRESUMO
In yeast, Rev1, Rev3, and Rev7 are involved in translesion synthesis over various kinds of DNA damage and spontaneous and UV-induced mutagenesis. Here, we disrupted Rev1, Rev3, and Rev7 in the chicken B-lymphocyte line DT40. REV1-/- REV3-/- REV7-/- cells showed spontaneous cell death, chromosomal instability/fragility, and hypersensitivity to various genotoxic treatments as observed in each of the single mutants. Surprisingly, the triple-knockout cells showed a suppressed level of sister chromatid exchanges (SCEs), which may reflect postreplication repair events mediated by homologous recombination, while each single mutant showed an elevated SCE level. Furthermore, REV1-/- cells as well as triple mutants showed a decreased level of immunoglobulin gene conversion, suggesting participation of Rev1 in a recombination-based pathway. The present study gives us a new insight into cooperative function of three Rev molecules and the Polzeta (Rev3-Rev7)-independent role of Rev1 in vertebrate cells.
Assuntos
Reparo do DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/fisiologia , Recombinação Genética/genética , Animais , Linfócitos B/enzimologia , Linhagem Celular , Galinhas/genética , Dano ao DNA/genética , Genes de Imunoglobulinas/genética , Mutagênese , Mutação , FenótipoRESUMO
BRCA2 is a tumor suppressor gene that is linked to hereditary breast and ovarian cancer. Although the Brca2 protein participates in homologous DNA recombination (HR), its precise role remains unclear. From chicken DT40 cells, we generated BRCA2 gene-deficient cells which harbor a truncation at the 3' end of the BRC3 repeat (brca2tr). Comparison of the characteristics of brca2tr cells with those of other HR-deficient DT40 clones revealed marked similarities with rad51 paralog mutants (rad51b, rad51c, rad51d, xrcc2, or xrcc3 cells). The phenotypic similarities include a shift from HR-mediated diversification to single-nucleotide substitutions in the immunoglobulin variable gene segment and the partial reversion of this shift by overexpression of Rad51. Although recent evidence supports at least Xrcc3 and Rad51C playing a role late in HR, our data suggest that Brca2 and the Rad51 paralogs may also contribute to HR at the same early step, with their loss resulting in the stimulation of an alternative, error-prone repair pathway.
Assuntos
Proteína BRCA2/metabolismo , Proteínas de Ligação a DNA/metabolismo , Região Variável de Imunoglobulina/genética , Mutação/genética , Recombinação Genética/genética , Animais , Proteínas Aviárias , Proteína BRCA2/genética , Células Cultivadas , Galinhas , Proteínas de Ligação a DNA/genética , Conversão Gênica/genética , Região Variável de Imunoglobulina/imunologia , Rad51 RecombinaseRESUMO
Homologous recombination (HR) requires nuclease activities at multiple steps, but the contribution of individual nucleases to the processing of double-strand DNA ends at different stages of HR has not been clearly defined. We used chicken DT40 cells to investigate the role of flap endonuclease 1 (Fen-1) in HR. FEN-1-deficient cells exhibited a significant decrease in the efficiency of immunoglobulin gene conversion while being proficient in recombination between sister chromatids, suggesting that Fen-1 may play a role in HR between sequences of considerable divergence. To clarify whether sequence divergence at DNA ends is truly the reason for the observed HR defect in FEN-1(-/-) cells we inserted a unique I-SceI restriction site in the genome and tested various donor and recipient HR substrates. We found that the efficiency of HR-mediated DNA repair was indeed greatly diminished when divergent sequences were present at the DNA break site. We conclude that Fen-1 eliminates heterologous sequences at DNA damage site and facilitates DNA repair by HR.
Assuntos
Dano ao DNA , Endonucleases Flap/fisiologia , Recombinação Genética , Animais , Sequência de Bases , Ciclo Celular , Galinhas , DNA/química , Reparo do DNA , DNA Complementar/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia , Endonucleases Flap/metabolismo , Citometria de Fluxo , Raios gama , Cinética , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência do Ácido Nucleico , Troca de Cromátide Irmã , Fatores de Tempo , TransfecçãoRESUMO
DNA double strand break (DSB) is one of the most critical types of damage which is induced by ionizing radiation. In this review, we summarize current progress in investigations on the function of DSB repair-related proteins. We focused on recent findings in the analysis of the function of proteins such as 53BP1, histone H2AX, Mus81-Eme1, Fanc complex, and UBC13, which are found to be related to homologous recombination repair or to non-homologous end joining. In addition to the function of these proteins in DSB repair, the biological function of nuclear foci formation following DSB induction is discussed.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Histonas/fisiologia , Humanos , Proteínas Nucleares/fisiologia , Transdução de Sinais/fisiologia , Enzimas de Conjugação de Ubiquitina/fisiologiaRESUMO
Nitric oxide (NO), a signal transmitter involved in inflammation and regulation of smooth muscle and neurons, seems to cause mutagenesis, but its mechanisms have remained elusive. To gain an insight into NO-induced genotoxicity, we analyzed the effect of NO on a panel of chicken DT40 clones deficient in DNA repair pathways, including base and nucleotide excision repair, double-strand break repair, and translesion DNA synthesis (TLS). Our results show that cells deficient in Rev1 and Rev3, a subunit essential for DNA polymerase zeta (Polzeta), are hypersensitive to killing by two chemical NO donors, spermine NONOate and S-nitroso-N-acetyl-penicillamine. Mitotic chromosomal analysis indicates that the hypersensitivity is caused by a significant increase in the level of induced chromosomal breaks. The data reveal the critical role of TLS polymerases in cellular tolerance to NO-induced DNA damage and suggest the contribution of these error-prone polymerases to accumulation of single base substitutions.
Assuntos
Dano ao DNA , Óxido Nítrico/toxicidade , Nucleotidiltransferases/fisiologia , Animais , Técnicas de Cultura de Células , Galinhas , Aberrações Cromossômicas , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Mutação PuntualRESUMO
Repair of DNA double strand breaks (DSBs) plays a critical role in the maintenance of the genome. DSB arise frequently as a consequence of replication fork stalling and also due to the attack of exogenous agents. Repair of broken DNA is essential for survival. Two major pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to deal with these lesions, and are conserved from yeast to vertebrates. Despite the conservation of these pathways, their relative contribution to DSB repair varies greatly between these two species. HR plays a dominant role in any DSB repair in yeast, whereas NHEJ significantly contributes to DSB repair in vertebrates. This active NHEJ requires a regulatory mechanism to choose HR or NHEJ in vertebrate cells. In this review, we illustrate how HR and NHEJ are differentially regulated depending on the phase of cell cycle and on the nature of the DSB.
Assuntos
Quebra Cromossômica , Reparo do DNA , Replicação do DNA , Recombinação Genética , Saccharomyces cerevisiae/genética , Animais , Proteínas de Ligação a DNA/genética , Humanos , Modelos Genéticos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In Saccharomyces cerevisiae, Rad18 functions in post-replication repair pathways, such as error-free damage bypass involving Rad30 (Poleta) and error-prone damage bypass involving Rev3/7 (Polzeta). Chicken DT40 RAD18(-/-) cells were found to be hypersensitive to camptothecin (CPT), while RAD30(-/-) and REV3(-/-) cells, which are defective in translesion DNA synthesis, were not. RAD18(-/-) cells also showed higher levels of H2AX phosphorylation and chromosomal aberrations, particularly chromosomal gaps and breaks, upon exposure to CPT. Detailed analysis by alkaline sucrose density gradient centrifugation revealed that RAD18(-/-) and wild type cells exhibited similar rates of elongation of newly synthesized DNA in the presence or absence of low concentrations of CPT but that DNA breaks frequently occurred on both parental and nascent strands within 1h after a brief exposure to an elevated concentration of CPT, with more breaks induced in RAD18(-/-) cells than in wild type cells. These data suggest a previously unanticipated role for Rad18 in dealing with replication forks upon encountering DNA lesions induced by CPT.
Assuntos
Camptotecina/toxicidade , Dano ao DNA , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Animais , Linhagem Celular , Galinhas/genética , Galinhas/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/fisiologia , GenomaRESUMO
Metazoan Rad51 plays a central role in homologous DNA recombination, and its activity is controlled by a number of Rad51 cofactors. These include five Rad51 paralogs, Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3. We previously hypothesized that all five paralogs participate collaboratively in repair. However, this idea was challenged by the biochemical identification of two independent complexes composed of either Rad51B/C/D/XRCC2 or Rad51C/XRCC3. To investigate if this biochemical finding is matched by genetic interactions, we made double mutants in either the same complex (rad51b/rad51d) or in both complexes (xrcc3/rad51d). In agreement with the biochemical findings the double deletion involving both complexes had an additive effect on the sensitivity to camptothecin and cisplatin. The double deletion of genes in the same complex, on the other hand, did not further increase the sensitivity to these agents. Conversely, all mutants tested displayed comparatively mild sensitivity to gamma-irradiation and attenuated gamma-irradiation-induced Rad51 foci formation. Thus, in accord with our previous conclusion, all paralogs appear to collaboratively facilitate Rad51 action. In conclusion, our detailed genetic study reveals a complex interplay between the five Rad51 paralogs and suggests that some of the Rad51 paralogs can separately operate in later step of homologous recombination.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Recombinação Genética , Animais , Camptotecina/toxicidade , Linhagem Celular , Cromátides , Quebra Cromossômica , Cisplatino/toxicidade , Replicação do DNA , Proteínas de Ligação a DNA/genética , Deleção de Genes , Camundongos , Radiação IonizanteRESUMO
Synchronization of cells is essential to study cell cycle specific events. If, for example, one suspects that a given DNA repair pathway is used in a particular cell cycle phase, the protocol can be used to enrich cells in each phase of the cell cycle and analyze the cellular response to DNA damage. Synchronization is also useful, when a gene is essential for a particular phase of the cell cycle. If a gene is, for example, essential for mitosis, synchronization of the cells in G1 phase with concomitant inactivation of the gene enables us to study the function of the gene in interphase, and to follow synchronous cell cycle progression to M phase. Two synchronization methods: centrifugal elutriation to enrich G1, S or G2 phase cells and nocodazole-mimocine sequential treatment to enrich cells at the G1/S boundary are described. Centrifugal elutriation can be achieved in less time (0.5-2 h) and with very little physiological stress to the cells whereas synchronization by drugs, such as nocodazole and mimocine, may result in unfavorable side effects.