RESUMO
Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA Helicases/metabolismo , Proteínas dos Microfilamentos/metabolismo , Processamento de Proteína Pós-Traducional , Translocação Genética , ATPases Associadas a Diversas Atividades Celulares , Linhagem Celular , Reparo do DNA , Proteínas de Ligação a DNA , Etoposídeo/toxicidade , Humanos , Fosforilação , Rad51 Recombinase/metabolismoRESUMO
Deficiency of X-ray repair cross-complementing protein 3 (XRCC3), a DNA-damage repair molecule, and the 241Met variant of XRCC3 have been reported to increase endoreduplication, which induces polyploidy. The aims of this study were to determine the impact of the XRCC3 polymorphism on the incidence of hypertension-induced left ventricular hypertrophy (LVH) and to investigate the mechanisms underlying any potential relationship. Patients undergoing chronic hemodialysis (n = 77) were genotyped to assess for the XRCC3 Thr241Met polymorphism. The XRCC3 241Thr/Met genotype was more frequent in the LVH (+) group than in the LVH (-) group (42.3 vs. 13.7%, χ2 = 7.85, p = 0.0051). To investigate possible mechanisms underlying these observations, human XRCC3 cDNA of 241Thr or that of 241Met was introduced into cultured CHO cells. The surface area of CHO cells expressing XRCC3 241Met was larger than that expressing 241Thr. Spontaneous DNA double-strand breaks accumulated to a greater degree in NIH3T3 cells expressing 241Met (3T3-241Met) than in those expressing 241Thr (3T3-241Thr). DNA damage caused by radiation induced cell senescence more frequently in 3T3-241Met. The levels of basal and TNF-α-stimulated MCP-1 mRNA and protein secretion were higher in 3T3-241Met. Finally, FACS analysis revealed that the cell percentage in G2/M phase including polyploidy was significantly higher in 3T3-241Met than in 3T3-241Thr. Furthermore, the basal level of MCP-1 mRNA positively correlated with the cell percentage in G2/M phase and polyploidy. These data suggest that the XRCC3 241Met increases the risk of LVH via accumulation of DNA damage, thereby altering cell cycle progression and inducing cell senescence and a proinflammatory phenotype.
Assuntos
Proteínas de Ligação a DNA/genética , Hipertensão/complicações , Hipertrofia Ventricular Esquerda/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Células CHO , Cricetulus , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Células NIH 3T3RESUMO
The epidermal growth factor receptor (EGFR) tyrosine kinase signaling pathways regulate cellular activities. The EGFR tyrosine kinase inhibitors (EGFR-TKIs) repress the EGFR pathway constitutively activated by somatic EGFR gene mutations and have drastically improved the prognosis of non-small-cell lung cancer (NSCLC) patients. However, some problems, including resistance, remain to be solved. Recently, combination therapy with EGFR-TKIs and cytotoxic agents has been shown to improve the prognosis of NSCLC patients. To enhance the anticancer effects of EGFR-TKIs, we examined the cross-talk of the EGFR pathways with ataxia telangiectasia-mutated (ATM) signaling pathways. ATM is a key protein kinase in the DNA damage response and is known to phosphorylate Akt, an EGFR downstream factor. We found that the combination of an ATM inhibitor, KU55933, and an EGFR-TKI, gefitinib, resulted in synergistic cell growth inhibition and induction of apoptosis in NSCLC cell lines carrying the sensitive EGFR mutation. We also found that KU55933 enhanced the gefitinib-dependent repression of the phosphorylation of EGFR and/or its downstream factors. ATM inhibition may facilitate the gefitinib-dependent repression of the phosphorylation of EGFR and/or its downstream factors, to exert anticancer effects against NSCLC cells with the sensitive EGFR mutation.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Humanos , Morfolinas/administração & dosagem , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Pironas/administração & dosagem , Quinazolinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: The reorganization of damaged chromatin plays an important role in the regulation of the DNA damage response. A recent study revealed the presence of 2 vertebrate H2A.Z isoforms, H2A.Z-1 and H2A.Z-2. However, the roles of the vertebrate H2A.Z isoforms are still unclear. Thus, in this study we examined the roles of the vertebrate H2A.Z isoforms in chromatin reorganization after the induction of DNA double-strand breaks (DSBs). METHODS AND MATERIALS: To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing each of the green fluorescent protein (GFP)-labeled H2A.Z isoforms, and performed fluorescence recovery after photobleaching (FRAP) analysis and inverted FRAP analysis in combination with microirradiation. Immunofluorescence staining using an anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2-Gy irradiation of wild-type (WT), H2A.Z-1- and H2A.Z-2-deficient DT40 cells. Colony-forming assays were also performed to compare the survival rates of WT, H2A.Z-1-, and H2A.Z-2-deficient DT40 cells with control, and H2A.Z-1- and H2A.Z-2-depleted U2OS cells after irradiation. RESULTS: FRAP analysis revealed that H2A.Z-2 was incorporated into damaged chromatin just after the induction of DSBs, whereas H2A.Z-1 remained essentially unchanged. Inverted FRAP analysis showed that H2A.Z-2 was released from damaged chromatin. These findings indicated that H2A.Z-2 was exchanged at DSB sites immediately after the induction of DSBs. RAD51 focus formation after ionizing irradiation was disturbed in H2A.Z-2-deficient DT40 cells but not in H2A.Z-1-deficient cells. The survival rate of H2A.Z-2-deficient cells after irradiation was lower than those of WT and H2A.Z-1- DT40 cells. Similar to DT40 cells, H2A.Z-2-depleted U2OS cells were also radiation-sensitive compared to control and H2A.Z-1-depleted cells. CONCLUSIONS: We found that vertebrate H2A.Z-2 is involved in the regulation of the DNA damage response at a very early stage, via the damaged chromatin reorganization required for RAD51 focus formation.
Assuntos
Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Histonas/metabolismo , Rad51 Recombinase/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Cromatina/química , Cromatina/genética , Ensaio de Unidades Formadoras de Colônias/métodos , Imunofluorescência/métodos , Histonas/genética , Humanos , Isoformas de Proteínas/metabolismoRESUMO
Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.
Assuntos
Cromatina/metabolismo , DNA Helicases/metabolismo , Histonas/metabolismo , Recombinação Homóloga/genética , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Rad51 Recombinase/metabolismo , Adenosina Trifosfatases/metabolismo , Linhagem Celular , Cromatina/genética , DNA Helicases/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Histonas/genética , Humanos , Proteínas Nucleares/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Rad51 Recombinase/genética , tRNA MetiltransferasesRESUMO
The analysis of dicentric chromosomes in human peripheral blood lymphocytes (PBLs) by Giemsa staining is the most established method for biological dosimetry. However, this method requires a well-trained person because of the difficulty in detecting aberrations rapidly and accurately. Here, we applied a fluorescence in situ hybridization (FISH) technique, using telomere and centromere peptide nucleic acid (PNA) probes, to solve the problem of biological dosimetry in radiation emergency medicine. A comparison by a well-trained observer found that FISH analysis of PBLs for the dose estimation was more accurate than the conventional Giemsa analysis, especially in samples irradiated at high doses. These results show that FISH analysis with centromeric/telomeric PNA probes could become the standard method for biological dosimetry in radiation emergency medicine.
Assuntos
Aberrações Cromossômicas/efeitos da radiação , Cromossomos Humanos/efeitos da radiação , Hibridização in Situ Fluorescente/métodos , Sondas Moleculares , Ácidos Nucleicos Peptídicos , Radiometria/métodos , Adulto , Corantes Azur , Centrômero/ultraestrutura , Quebra Cromossômica/efeitos da radiação , Cromossomos Humanos/ultraestrutura , Relação Dose-Resposta à Radiação , Medicina de Emergência/métodos , Feminino , Raios gama/efeitos adversos , Humanos , Técnicas In Vitro , Linfócitos/efeitos da radiação , Linfócitos/ultraestrutura , Masculino , Metáfase , Pessoa de Meia-Idade , Ácidos Nucleicos Peptídicos/genética , Cromossomos em Anel , Coloração e Rotulagem , Telômero/ultraestruturaRESUMO
The meiosis-specific synaptonemal complex protein SYCP3 has been reported to be aberrantly expressed in tumours. However, in contrast to its well-defined function in meiosis, its possible role in mitotic cells is entirely unknown. Here, we show that SYCP3 is expressed in a range of primary tumours and that it impairs chromosomal integrity in mitotic cells. Expression of SYCP3 inhibits the homologous recombination (HR) pathway mediated by RAD51, inducing hypersensitivity to DNA-damaging agents such as a poly(ADP-ribose) polymerase (PARP) inhibitor and chromosomal instability. SYCP3 forms a complex with BRCA2 and inhibits its role in HR. These findings highlight a new mechanism for chromosomal instability in cancer and extend the range of PARP-inhibitor sensitive tumours to those expressing SYCP3.
Assuntos
Proteína BRCA2/metabolismo , Recombinação Homóloga , Mitose/genética , Proteínas Nucleares/metabolismo , Aneuploidia , Proteínas de Ciclo Celular , Instabilidade Cromossômica , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA , Resistencia a Medicamentos Antineoplásicos/genética , Inativação Gênica , Células Hep G2 , Humanos , Proteínas Nucleares/genética , Inibidores de Poli(ADP-Ribose) Polimerases , Ligação Proteica , Rad51 Recombinase/metabolismo , Tolerância a Radiação/genética , Troca de Cromátide IrmãRESUMO
Defects in the FANCJ/BRIP1 helicase gene are associated with genome instability disorders such as familial breast cancer or Fanconi anemia (FA). Although FANCJ has an in vitro activity to resolve G-quadruplex (G4) structures, and FANCJ ortholog in C. elegans prevents G4-associated deletions during replication, how FANCJ loss affects genome integrity in higher organisms remains unclear. Here, we report that FANCJ, but not other FA genes FANCD2 or FANCC, protected against large-scale genomic deletion that occurred frequently at the rearranged immunoglobulin heavy chain (IgH) locus in chicken DT40 cell line, suggesting that FancJ protects the genome independently of the FA ubiquitination pathway. In a more unbiased approach using array-comparative genomic hybridization, we identified de novo deletions as well as amplifications in fancj cells kept in culture for 2 months. A cluster of G4 sequence motifs was found near the breakpoint of one amplified region, but G4 sequence motifs were not detected at the breakpoints of two deleted regions. These results collectively suggest that, unlike in C. elegans, actions of vertebrate FANCJ to promote genome stability may not be limited to protection against the G4-mediated gene deletions.
Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Instabilidade Genômica/genética , RNA Helicases/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Galinhas , Hibridização Genômica Comparativa , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Quadruplex G , Amplificação de Genes/genética , Conversão Gênica/genética , Deleção de Genes , Ordem dos Genes , Rearranjo Gênico/genética , Marcação de Genes , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Dados de Sequência Molecular , Nucleosídeo Desaminases/genética , Nucleosídeo Desaminases/metabolismo , RNA Helicases/genética , Alinhamento de SequênciaRESUMO
Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Recombinação Genética , Translocação Genética , Proteínas Supressoras de Tumor/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Cromossomos Humanos Par 11 , Proteínas de Ligação a DNA/genética , Etoposídeo/farmacologia , Histona-Lisina N-Metiltransferase , Humanos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Rad51C is a central component of two complexes formed by five Rad51 paralogs in vertebrates. These complexes are involved in repairing DNA double-strand breaks through homologous recombination. Despite accumulating evidence suggesting that the paralogs may prevent aneuploidy by controlling centrosome integrity, Rad51C's role in maintaining chromosome stability remains unclear. Here we demonstrate that Rad51C deficiency leads to both centrosome aberrations in an ATR-Chk1-dependent manner and increased aneuploidy in human cells. While it was reported that Rad51C deficiency did not cause centrosome aberrations in interphase in hamster cells, such aberrations were observed in interphase in HCT116 cells with Rad51C dysfunction. Caffeine treatment and down-regulation of ATR, but not that of ATM, reduced the frequency of centrosome aberrations in the mutant cells. Silencing of Rad51C by RNA interference in HT1080 cells resulted in similar aberrations. Treatment with a Chk1 inhibitor and silencing of Chk1 also reduced the frequency in HCT116 mutants. Accumulation of Chk1 at the centrosome and nuclear foci of gamma H2AX were increased in the mutants. Moreover, the mutant cells had a higher frequency of aneuploidy. These findings indicate that the ATR-Chk1 pathway plays a role in increased centrosome aberrations induced by Rad51C dysfunction.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aneuploidia , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Humanos , Interferência de RNA , Recombinação GenéticaRESUMO
The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response.
Assuntos
Nucléolo Celular/química , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Resposta ao Choque Térmico , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Processamento Alternativo , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/química , Galinhas , Inibidores de Cisteína Proteinase/farmacologia , Citoplasma/química , Proteínas de Ligação a DNA/química , Dactinomicina/farmacologia , Éxons , Deleção de Genes , Humanos , Dados de Sequência Molecular , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores de Proteassoma , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The Rad51-like proteins, Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3, have been shown to form two distinct complexes and seem to assist Rad51 in the early stages of homologous recombination. Although these proteins share sequence similarity with Rad51, they do not show functional redundancy. Among them, Rad51B is unique in that the gene maps to the human chromosome 14q23-24, the region frequently involved in balanced chromosome translocations in benign tumors particularly in uterine leiomyomas. Despite accumulating descriptive evidence of altered Rad51B function in these tumors, the biological significance of this aberration is still unknown. To assess the significance of reduced Rad51B function, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here, we show that haploinsufficiency of RAD51B causes mild hypersensitivity to DNA-damaging agents, a mild reduction in sister chromatid exchange, impaired Rad51 focus formation, and an increase in chromosome aberrations. Remarkably, haploinsufficiency of RAD51B leads to centrosome fragmentation and aneuploidy. In addition, an approximately 50% reduction in RAD51B mRNA levels by RNA interference also leads to centrosome fragmentation in the human fibrosarcoma cell line HT1080. These findings suggest that the proper biallelic expression of RAD51B is required for the maintenance of chromosome integrity in human cells.
Assuntos
Aneuploidia , Centrossomo/metabolismo , Neoplasias do Colo/genética , Proteínas de Ligação a DNA/genética , Aberrações Cromossômicas , Neoplasias do Colo/metabolismo , Dano ao DNA , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Marcação de Genes , Células HCT116 , Haploidia , Humanos , RNA Interferente Pequeno/genética , Troca de Cromátide IrmãRESUMO
The Mus81-Eme1 complex is a structure-specific endonuclease that preferentially cleaves nicked Holliday junctions, 3'-flap structures and aberrant replication fork structures. Mus81-/- mice have been shown to exhibit spontaneous chromosomal aberrations and, in one of two models, a predisposition to cancers. The molecular mechanisms underlying its role in chromosome integrity, however, are largely unknown. To clarify the role of Mus81 in human cells, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here we demonstrate that Mus81 confers resistance to DNA crosslinking agents and slight resistance to other DNA-damaging agents. Mus81 deficiency spontaneously promotes chromosome damage such as breaks and activates the intra-S-phase checkpoint through the ATM-Chk1/Chk2 pathways. Furthermore, Mus81 deficiency activates the G2/M checkpoint through the ATM-Chk2 pathway and promotes DNA rereplication. Increased rereplication is reversed by the ectopic expression of Cdk1. Haploinsufficiency of Mus81 or Eme1 also causes similar phenotypes. These findings suggest that a complex network of the checkpoint pathways that respond to DNA double-strand breaks may participate in some of the phenotypes associated with Mus81 or Eme1 deficiency.
Assuntos
Ciclo Celular , Instabilidade Cromossômica , Proteínas de Ligação a DNA/fisiologia , Endodesoxirribonucleases/fisiologia , Endonucleases/fisiologia , Poliploidia , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Fase G2 , Deleção de Genes , Marcação de Genes , Heterozigoto , Humanos , Mitose , Fase SRESUMO
XRCC3 was inactivated in human cells by gene targeting. Consistent with its role in homologous recombination, XRCC3(-/-) cells showed a two-fold sensitivity to DNA cross-linking agents, a mild reduction in sister chromatid exchange, impaired Rad51 focus formation and elevated chromosome aberrations. Furthermore, endoreduplication was increased five- seven-fold in the mutants. The T241M variant of XRCC3 has been associated with an increased cancer risk. Expression of the wild-type cDNA restored this phenotype, while expression of the variant restored the defective recombinational repair, but not the increased endoreduplication. RPA, a protein essential for homologous recombination and DNA replication, is associated with XRCC3 and Rad52. Overexpression of RPA promoted endoreduplication, which was partially complemented by overexpression of the wild-type XRCC3 protein, but not by overexpression of the variant protein. Overexpression of Rad52 prevented endoreduplication in RPA-overexpressing cells, in XRCC3(-/-) cells and in the variant-expressing cells, suggesting that deregulated RPA was responsible for the increased endoreduplication. These observations offer the first genetic evidence for the association between homologous recombination and replication initiation having a role in cancer susceptibility.
Assuntos
Agmatina/análogos & derivados , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Ploidias , Recombinação Genética , Agmatina/farmacologia , Linhagem Celular , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Deleção de Genes , Expressão Gênica , Marcação de Genes , Humanos , Mitomicina/farmacologia , Proteína Rad52 de Recombinação e Reparo de DNA , Proteína de Replicação A , Succinatos/farmacologiaRESUMO
In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.