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1.
Proc Natl Acad Sci U S A ; 117(28): 16527-16536, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601218

RESUMO

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.


Assuntos
Endodesoxirribonucleases/metabolismo , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitose , Rad51 Recombinase/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo
2.
Nucleic Acids Res ; 45(16): 9427-9440, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28934474

RESUMO

DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display a mild mutator phenotype, while nuclear extracts of these cells exhibit reduced MMR activity in vitro, and these defects are complemented by overexpression or addition of exogenous human Exonuclease 1 (EXO1). By contrast, another proofreading-deficient mutant, PolδD515V, which has a weaker strand displacement activity, does not decrease the MMR activity as significantly as PolδD316A;E318A. In addition, PolδD515V does not increase the mutation frequency in MMR-proficient cells. Based on our findings, we propose that the proofreading activity restricts the strand displacement activity of Polδ in MMR. This contributes to maintain the nicks required for EXO1 entry, and in this manner ensures the dominance of the EXO1-dependent MMR pathway.


Assuntos
Reparo de Erro de Pareamento de DNA , DNA Polimerase III/metabolismo , Mutação , Metilação de DNA/efeitos dos fármacos , DNA Polimerase III/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Células HeLa , Humanos , Metilnitronitrosoguanidina/farmacologia
4.
Cell Oncol (Dordr) ; 40(4): 341-355, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28536927

RESUMO

BACKGROUND: Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. METHODS: The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 µM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined. RESULTS: MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p < 0.05) lower expression of the MMR and pluripotency factors, as well as a reduced MMR activity and cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p < 0.001) reduced cisplatin sensitivity. CONCLUSION: This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced sensitivity to cisplatin in differentiated TGCT components could result from a reduced expression of MMR proteins, in particular MSH2 and MLH1, which are involved in the recognition of cisplatin adducts and in activation of the DNA damage response pathway to initiate apoptosis.


Assuntos
Cisplatino/uso terapêutico , Reparo de Erro de Pareamento de DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Neoplasias Testiculares/tratamento farmacológico , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Interferência de RNA , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia
5.
Nucleic Acids Res ; 44(6): 2691-705, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26743004

RESUMO

During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and -deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR.


Assuntos
Linfócitos B/metabolismo , Reparo de Erro de Pareamento de DNA/imunologia , DNA/genética , Switching de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/genética , Uracila/metabolismo , Linfócitos B/citologia , Linfócitos B/imunologia , Linhagem Celular Tumoral , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Citosina/imunologia , Citosina/metabolismo , DNA/imunologia , Quebras de DNA de Cadeia Dupla , Regulação da Expressão Gênica , Células HEK293 , Humanos , Transdução de Sinais , Uracila/imunologia , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/imunologia
6.
DNA Repair (Amst) ; 38: 147-154, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26708048

RESUMO

The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind by the replicative polymerases and results in increased mutation load at the genome. The realization that defective MMR leads to a hypermutation phenotype and increased risk of tumorigenesis highlights the relevance of this pathway for human disease. The association of MMR defects with increased risk of cancer development was first observed in colorectal cancer patients that carried inactivating germline mutations in MMR genes and the disease was named as hereditary non-polyposis colorectal cancer (HNPCC). Currently, a growing list of cancers is found to be MMR defective and HNPCC has been renamed Lynch syndrome (LS) partly to include the associated risk of developing extra-colonic cancers. In addition, a number of non-hereditary, mostly epigenetic, alterations of MMR genes have been described in sporadic tumors. Besides conferring a strong cancer predisposition, genetic or epigenetic inactivation of MMR genes also renders cells resistant to some chemotherapeutic agents. Therefore, diagnosis of MMR deficiency has important implications for the management of the patients, the surveillance of their relatives in the case of LS and for the choice of treatment. Some of the alterations found in MMR genes have already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR genes and anticipates the need of diagnostic tools for rapid assessment of their pathogenicity. This review describes current tools and future strategies for addressing the relevance of MMR gene alterations in human disease.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Mutação/genética , Neoplasias/diagnóstico , Neoplasias/genética , Pesquisa Biomédica , DNA/metabolismo , Humanos , Estabilidade Proteica
7.
Front Genet ; 5: 287, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25191341

RESUMO

DNA is constantly under attack by a number of both exogenous and endogenous agents that challenge its integrity. Among the mechanisms that have evolved to counteract this deleterious action, mismatch repair (MMR) has specialized in removing DNA biosynthetic errors that occur when replicating the genome. Malfunction or inactivation of this system results in an increase in spontaneous mutability and a strong predisposition to tumor development. Besides this key corrective role, MMR proteins are involved in other pathways of DNA metabolism such as mitotic and meiotic recombination and processing of oxidative damage. Surprisingly, MMR is also required for certain mutagenic processes. The mutagenic MMR has beneficial consequences contributing to the generation of a vast repertoire of antibodies through class switch recombination and somatic hypermutation processes. However, this non-canonical mutagenic MMR also has detrimental effects; it promotes repeat expansions associated with neuromuscular and neurodegenerative diseases and may contribute to cancer/disease-related aberrant mutations and translocations. The reaction responsible for replication error correction has been the most thoroughly studied and it is the subject to numerous reviews. This review describes briefly the biochemistry of MMR and focuses primarily on the non-canonical MMR activities described in mammals as well as emerging research implicating interplay of MMR and chromatin.

8.
Nucleic Acids Res ; 41(5): 2846-56, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23325852

RESUMO

Genome-wide gene expression analyses of the human somatic cell cycle have indicated that the set of cycling genes differ between primary and cancer cells. By identifying genes that have cell cycle dependent expression in HaCaT human keratinocytes and comparing these with previously identified cell cycle genes, we have identified three distinct groups of cell cycle genes. First, housekeeping genes enriched for known cell cycle functions; second, cell type-specific genes enriched for HaCaT-specific functions; and third, Polycomb-regulated genes. These Polycomb-regulated genes are specifically upregulated during DNA replication, and consistent with being epigenetically silenced in other cell cycle phases, these genes have lower expression than other cell cycle genes. We also find similar patterns in foreskin fibroblasts, indicating that replication-dependent expression of Polycomb-silenced genes is a prevalent but unrecognized regulatory mechanism.


Assuntos
Ciclo Celular/genética , Replicação do DNA , Proteínas do Grupo Polycomb/fisiologia , Regulação para Cima , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Ilhas de CpG , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Genes Essenciais , Histonas/fisiologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Análise dos Mínimos Quadrados , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética , Transcriptoma
9.
Mol Cell ; 47(5): 669-80, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22864113

RESUMO

Mismatch repair (MMR) is a key antimutagenic process that increases the fidelity of DNA replication and recombination. Yet genetic experiments showed that MMR is required for antibody maturation, a process during which the immunoglobulin loci of antigen-stimulated B cells undergo extensive mutagenesis and rearrangements. In an attempt to elucidate the mechanism underlying the latter events, we set out to search for conditions that compromise MMR fidelity. Here, we describe noncanonical MMR (ncMMR), a process in which the MMR pathway is activated by various DNA lesions rather than by mispairs. ncMMR is largely independent of DNA replication, lacks strand directionality, triggers PCNA monoubiquitylation, and promotes recruitment of the error-prone polymerase-η to chromatin. Importantly, ncMMR is not limited to B cells but occurs also in other cell types. Moreover, it contributes to mutagenesis induced by alkylating agents. Activation of ncMMR may therefore play a role in genomic instability and cancer.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Instabilidade Genômica/genética , Células Cultivadas , Replicação do DNA , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo
10.
Trends Biochem Sci ; 37(5): 206-14, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22475811

RESUMO

A considerable surge of interest in the mismatch repair (MMR) system has been brought about by the discovery of a link between Lynch syndrome, an inherited predisposition to cancer of the colon and other organs, and malfunction of this key DNA metabolic pathway. This review focuses on recent advances in our understanding of the molecular mechanisms of canonical MMR, which improves replication fidelity by removing misincorporated nucleotides from the nascent DNA strand. We also discuss the involvement of MMR proteins in two other processes: trinucleotide repeat expansion and antibody maturation, in which MMR proteins are required for mutagenesis rather than for its prevention.


Assuntos
Pareamento Incorreto de Bases/genética , Reparo de Erro de Pareamento de DNA , Replicação do DNA/genética , Mamíferos/genética , Animais , DNA/genética , Humanos , Modelos Genéticos , Expansão das Repetições de Trinucleotídeos/genética
11.
Environ Mol Mutagen ; 52(8): 623-35, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21786338

RESUMO

XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLß and PNK colocalize with XRCC1 in replication foci and that POLß and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLß and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLß to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes.


Assuntos
Quebras de DNA de Cadeia Simples , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Animais , Western Blotting , Células CHO , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Quebras de DNA de Cadeia Simples/efeitos da radiação , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta à Radiação , Células HeLa , Humanos , Imunoprecipitação , Lasers , Microscopia Confocal , Modelos Biológicos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Polinucleotídeo 5'-Hidroxiquinase/genética , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
12.
EMBO Rep ; 11(12): 962-8, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21052091

RESUMO

End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1). In this study, we show that the localization of EXO1 to DSBs depends on both CtIP and MRN. We also establish that CtIP interacts with EXO1 and restrains its exonucleolytic activity in vitro. Finally, we show that on exposure to camptothecin, depletion of EXO1 in CtIP-deficient cells increases the frequency of DNA-PK-dependent radial chromosome formation. Thus, our study identifies new functions of CtIP and EXO1 in DNA end resection and provides new information on the regulation of DSB repair pathways, which is a key factor in the maintenance of genome integrity.


Assuntos
Proteínas de Transporte/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Instabilidade Genômica , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Citoproteção , Quebras de DNA de Cadeia Dupla , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Células HEK293 , Humanos , Proteína Homóloga a MRE11 , Ligação Proteica , Recombinação Genética/genética
14.
DNA Repair (Amst) ; 8(7): 834-43, 2009 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-19442590

RESUMO

Base excision repair (BER) of damaged or inappropriate bases in DNA has been reported to take place by single nucleotide insertion or through incorporation of several nucleotides, termed short-patch and long-patch repair, respectively. We found that extracts from proliferating and non-proliferating cells both had capacity for single- and two-nucleotide insertion BER activity. However, patch size longer than two nucleotides was only detected in extracts from proliferating cells. Relative to extracts from proliferating cells, extracts from non-proliferating cells had approximately two-fold higher concentration of POLbeta, which contributed to most of two-nucleotide insertion BER. In contrast, two-nucleotide insertion in extracts from proliferating cells was not dependent on POLbeta. BER fidelity was two- to three-fold lower in extracts from the non-proliferating compared with extracts of proliferating cells. Furthermore, although one-nucleotide deletion was the predominant type of repair error in both extracts, the pattern of repair errors was somewhat different. These results establish two-nucleotide patch BER as a distinct POLbeta-dependent mechanism in non-proliferating cells and demonstrate that BER fidelity is lower in extracts from non-proliferating as compared with proliferating cells.


Assuntos
Proliferação de Células , Reparo do DNA/fisiologia , Linfócitos/metabolismo , Transdução de Sinais/fisiologia , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Extratos Celulares/química , Linhagem Celular , Células Cultivadas , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Linfócitos/química , Linfócitos/citologia , Mutação , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Especificidade por Substrato
15.
DNA Repair (Amst) ; 8(7): 822-33, 2009 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-19411194

RESUMO

The PA promoter in the human uracil-DNA glycosylase gene (UNG) directs expression of the nuclear form (UNG2) of UNG proteins. Using a combination of promoter deletion and mutation analyses, and transient transfection of HeLa cells, we show that repressor and derepressor activities are contained within the region of DNA marked by PA. Footprinting analysis and electrophoretic mobility shift assays of PA and putative AP-2 binding regions with HeLa cell nuclear extract and recombinant AP-2alpha protein indicate that AP-2 transcription factors are central in the regulated expression of UNG2 mRNA. Chromatin immunoprecipitation with AP-2 antibody demonstrated that endogenous AP-2 binds to the PA promoter in vivo. Overexpression of AP-2alpha, -beta or -gamma all stimulated expression from a PA-luciferase reporter gene construct approximately 3- to 4-fold. Interestingly, an N-terminally truncated AP-2alpha, lacking the activation domain but retaining the DNA binding and dimerization domains, stimulated PA to a level approaching that of full-length AP-2, suggesting that AP-2 overexpression stimulates PA activity by a mechanism involving derepression rather than activation, possibly by neutralizing an inhibitory effect of endogenous AP-2 or AP-2-like factors.


Assuntos
Regulação Enzimológica da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição AP-2/metabolismo , Uracila-DNA Glicosidase/genética , Sequência de Bases , Sítios de Ligação/genética , Fator de Ligação a CCAAT/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Pegada de DNA , Desoxirribonuclease I/metabolismo , Fatores de Transcrição E2F/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição AP-2/genética , Transfecção , Tretinoína/farmacologia
16.
J Cell Sci ; 122(Pt 8): 1258-67, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19299466

RESUMO

RECQL4 belongs to the conserved RecQ family of DNA helicases, members of which play important roles in the maintenance of genome stability in all organisms that have been examined. Although genetic alterations in the RECQL4 gene are reported to be associated with three autosomal recessive disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes), the molecular role of RECQL4 still remains poorly understood. Here, we show that RECQL4 specifically interacts with the histone acetyltransferase p300 (also known as p300 HAT), both in vivo and in vitro, and that p300 acetylates one or more of the lysine residues at positions 376, 380, 382, 385 and 386 of RECQL4. Furthermore, we report that these five lysine residues lie within a short motif of 30 amino acids that is essential for the nuclear localization of RECQL4. Remarkably, the acetylation of RECQL4 by p300 in vivo leads to a significant shift of a proportion of RECQL4 protein from the nucleus to the cytoplasm. This accumulation of the acetylated RECQL4 is a result of its inability to be imported into the nucleus. Our results provide the first evidence of a post-translational modification of the RECQL4 protein, and suggest that acetylation of RECQL4 by p300 regulates the trafficking of RECQL4 between the nucleus and the cytoplasm.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteína p300 Associada a E1A/metabolismo , Processamento de Proteína Pós-Traducional , RecQ Helicases/metabolismo , Acetilação , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteína p300 Associada a E1A/genética , Células HeLa , Humanos , Lisina , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear/metabolismo , Transporte Proteico , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
17.
EMBO J ; 27(1): 51-61, 2008 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-18079698

RESUMO

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells.


Assuntos
Ciclo Celular/fisiologia , DNA Glicosilases/metabolismo , Proteína de Replicação A/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Bovinos , DNA Glicosilases/química , DNA Glicosilases/genética , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína de Replicação A/fisiologia , Serina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Treonina/metabolismo , Uracila/metabolismo
18.
Nucleic Acids Res ; 35(17): 5706-16, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17715146

RESUMO

Werner syndrome (WS) is a severe recessive disorder characterized by premature aging, cancer predisposition and genomic instability. The gene mutated in WS encodes a bi-functional enzyme called WRN that acts as a RecQ-type DNA helicase and a 3'-5' exonuclease, but its exact role in DNA metabolism is poorly understood. Here we show that WRN physically interacts with the MSH2/MSH6 (MutSalpha), MSH2/MSH3 (MutSbeta) and MLH1/PMS2 (MutLalpha) heterodimers that are involved in the initiation of mismatch repair (MMR) and the rejection of homeologous recombination. MutSalpha and MutSbeta can strongly stimulate the helicase activity of WRN specifically on forked DNA structures with a 3'-single-stranded arm. The stimulatory effect of MutSalpha on WRN-mediated unwinding is enhanced by a G/T mismatch in the DNA duplex ahead of the fork. The MutLalpha protein known to bind to the MutS alpha-heteroduplex complexes has no effect on WRN-mediated DNA unwinding stimulated by MutSalpha, nor does it affect DNA unwinding by WRN alone. Our data are consistent with results of genetic experiments in yeast suggesting that MMR factors act in conjunction with a RecQ-type helicase to reject recombination between divergent sequences.


Assuntos
Pareamento Incorreto de Bases , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , RecQ Helicases/metabolismo , Sítios de Ligação , Linhagem Celular , DNA/química , DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases , Humanos , Proteínas MutL , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS , Estrutura Terciária de Proteína , RecQ Helicases/química , Técnicas do Sistema de Duplo-Híbrido , Helicase da Síndrome de Werner
19.
Exp Cell Res ; 312(14): 2666-72, 2006 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16860315

RESUMO

Uracil is present in small amounts in DNA due to spontaneous deamination of cytosine and incorporation of dUMP during replication. While deamination generates mutagenic U:G mismatches, incorporated dUMP results in U:A pairs that are not directly mutagenic, but may be cytotoxic. In most cells, mutations resulting from uracil in DNA are prevented by error-free base excision repair. However, in B-cells uracil in DNA is also a physiological intermediate in acquired immunity. Here, activation-induced cytosine deaminase (AID) introduces template uracils that give GC to AT transition mutations in the Ig locus after replication. When uracil-DNA glycosylase (UNG2) removes uracil, error-prone translesion synthesis over the abasic site causes other mutations in the Ig locus. Together, these processes are central to somatic hypermutation (SHM) that increases immunoglobulin diversity. AID and UNG2 are also essential for generation of strand breaks that initiate class switch recombination (CSR). Patients lacking UNG2 display a hyper-IgM syndrome with recurrent infections, increased IgM, strongly decreased IgG, IgA and IgE and skewed SHM. UNG2 is also involved in innate immune response against retroviral infections. Ung(-/-) mice have a similar phenotype and develop B-cell lymphomas late in life. However, there is no evidence indicating that UNG deficiency causes lymphomas in humans.


Assuntos
DNA Glicosilases/genética , Reparo do DNA , DNA/química , Uracila/fisiologia , Animais , Humanos , Imunidade , Linfoma de Células B/genética , Camundongos , Modelos Genéticos , Hipermutação Somática de Imunoglobulina , Uracila-DNA Glicosidase/metabolismo
20.
DNA Repair (Amst) ; 4(12): 1432-41, 2005 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-16174566

RESUMO

Ung-deficient mice have reduced class switch recombination, skewed somatic hypermutation, lymphatic hyperplasia and a 22-fold increased risk of developing B-cell lymphomas. We find that lymphomas are of follicular (FL) and diffuse large B-cell type (DLBCL). All FLs and 75% of the DLBCLs were monoclonal while 25% were biclonal. Monoclonality was also observed in hyperplasia, and could represent an early stage of lymphoma development. Lymphoid hyperplasia occurs very early in otherwise healthy Ung-deficient mice, observed as a significant increase of splenic B-cells. Furthermore, loss of Ung also causes a significant reduction of T-helper cells, and 50% of the young Ung(-/-) mice investigated have no detectable NK/NKT-cell population in their spleen. The immunological imbalance is confirmed in experiments with spleen cells where the production of the cytokines interferon gamma, interleukin 6 and interleukin 2 is clearly different in wild type and in Ung-deficient mice. This suggests that Ung-proteins, directly or indirectly, have important functions in the immune system, not only in the process of antibody maturation, but also for production and functions of immunologically important cell types. The immunological imbalances shown here in the Ung-deficient mice may be central in the development of lymphomas in a background of generalised lymphoid hyperplasia.


Assuntos
Linfócitos B/patologia , Hiperplasia/patologia , Leucócitos/patologia , Linfoma de Células B/patologia , Linfócitos T/patologia , Uracila-DNA Glicosidase/deficiência , Animais , Concanavalina A/farmacologia , Citocinas/biossíntese , DNA/genética , Citometria de Fluxo , Perfilação da Expressão Gênica , Genótipo , Lectinas/farmacologia , Lipopolissacarídeos/farmacologia , Linfoma de Células B/diagnóstico , Camundongos , Baço/efeitos dos fármacos , Baço/patologia , Acetato de Tetradecanoilforbol/farmacologia , Uracila-DNA Glicosidase/genética
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