RESUMO
Werner syndrome (WS) is a premature aging disorder where the affected individuals appear much older than their chronological age. The single gene that is defective in WS encodes a protein (WRN) that has ATPase, helicase and 3'-->5' exonuclease activities. Our laboratory has recently uncovered a physical and functional interaction between WRN and the Ku heterodimer complex that functions in double-strand break repair and V(D)J recombination. Importantly, Ku specifically stimulates the exonuclease activity of WRN. We now report that Ku enables the Werner exonuclease to digest through regions of DNA containing 8-oxoadenine and 8-oxoguanine modifications, lesions that have previously been shown to block the exonuclease activity of WRN alone. These results indicate that Ku significantly alters the exonuclease function of WRN and suggest that the two proteins function concomitantly in a DNA damage processing pathway. In support of this notion we also observed co-localization of WRN and Ku, particularly after DNA damaging treatments.
Assuntos
Adenina/análogos & derivados , Antígenos Nucleares , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Exonucleases/metabolismo , Guanina/análogos & derivados , Proteínas Nucleares/metabolismo , Adenina/química , Linhagem Celular , Núcleo Celular/química , Dano ao DNA , DNA Helicases/análise , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/farmacologia , Proteínas de Ligação a DNA/fisiologia , Exodesoxirribonucleases , Exonucleases/análise , Exonucleases/fisiologia , Guanina/química , Células HeLa , Humanos , Autoantígeno Ku , Proteínas Nucleares/análise , Proteínas Nucleares/fisiologia , RecQ Helicases , Proteína de Replicação A , Helicase da Síndrome de WernerRESUMO
Werner syndrome is a premature aging syndrome displaying numerous signs and symptoms found in normal aging. The disease is associated with a mutation in the WRN gene. We have purified the Werner protein (WRN) and studied its biochemical activities and its protein interactions. WRN is a helicase and an exonuclease and also has an associated ATPase activity. WRN interacts physically and functionally with replication protein A (RPA), which stimulates its helicase activity. We have studied the WRN exonuclease activity and found that it can be blocked by certain DNA lesions and not by others. Thus, while WRN does not bind to DNA damage, it may have properties that allow it to sense the presence of damage in DNA. More recently we have found other protein interactions that involve physical and functional interactions with WRN, which could suggest a role for WRN in DNA repair.
Assuntos
Antígenos Nucleares , DNA Helicases/fisiologia , Síndrome de Werner/etiologia , Dano ao DNA , DNA Helicases/genética , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Exodesoxirribonucleases , Humanos , Autoantígeno Ku , Proteínas Nucleares/fisiologia , RecQ Helicases , Síndrome de Werner/genética , Helicase da Síndrome de WernerRESUMO
Ribosomal DNA (rDNA) metabolism has been implicated in cellular and organismal aging. The role of rDNA in premature and normal human aging was investigated by measuring rDNA gene copy number, the level of rDNA methylation, and rRNA expression during the in vitro senescence of primary fibroblasts from normal (young and old) donors and from Werner syndrome (WS) patients. In comparison to their normal counterparts, WS fibroblasts grew slowly and reached senescence after fewer doublings. The rDNA copy number did not change significantly throughout the life span of both normal and WS fibroblasts. However, in senescent WS and normal old fibroblasts, we detected rDNA species with unusually slow electrophoretic mobility. Cellular aging in Saccharomyces cerevisiae is accompanied by the formation and accumulation of rDNA circles. Our analysis revealed that the rDNA species observed in this study were longer, linear rDNA molecules attributable to the inhibition of ECO:RI cleavage by methylation. Furthermore, isoschizomeric restriction analysis confirmed that in vitro senescence of fibroblasts is accompanied by significant increases in cytosine methylation within rDNA genes. This increased methylation is maximal during the abbreviated life span of WS fibroblasts. Despite increased methylation of rDNA in senescent cells, the steady-state levels of 28S rRNA remained constant over the life span of both normal and WS fibroblasts.
Assuntos
RNA Ribossômico/genética , Síndrome de Werner/genética , Células Cultivadas , Senescência Celular/genética , Metilação de DNA , Fibroblastos/patologia , Dosagem de Genes , Humanos , RNA Ribossômico/metabolismo , RNA Ribossômico 28S/metabolismo , Síndrome de Werner/patologiaRESUMO
Individuals with mutations in the WRN gene suffer from Werner syndrome, a disease with early onset of many characteristics of normal aging. The WRN protein (WRNp) functions in DNA metabolism, as the purified polypeptide has both 3'-->5' helicase and 3'-->5' exonuclease activities. In this study, we have further characterized WRNp exonuclease activity by examining its ability to degrade double-stranded DNA substrates containing abnormal and damaged nucleo-tides. In addition, we directly compared the 3'-->5' WRNp exonuclease activity with that of exo-nuclease III and the Klenow fragment of DNA polymerase I. Our results indicate that the presence of certain abnormal bases (such as uracil and hypoxanthine) does not inhibit the exonuclease activity of WRNp, exo-nuclease III or Klenow, whereas other DNA modifications, including apurinic sites, 8-oxoguanine, 8-oxoadenine and cholesterol adducts, inhibit or block WRNp. The ability of damaged nucleo-tides to inhibit exonucleolytic digestion differs significantly between WRNp, exonuclease III and Klenow, indicating that each exonuclease has a distinct mechanism of action. In addition, normal and modified DNA substrates are degraded similarly by full-length WRNp and an N-terminal fragment of WRNp, indicating that the specificity for this activity lies mostly within this region. The biochemical and physiological significance of these results is discussed.
Assuntos
DNA Helicases/metabolismo , DNA/metabolismo , Exonucleases/metabolismo , Adenina/análogos & derivados , Adenina/química , Sequência de Bases , DNA/química , DNA/genética , Adutos de DNA/química , Adutos de DNA/metabolismo , DNA Helicases/genética , DNA Polimerase I/metabolismo , Exodesoxirribonucleases/metabolismo , Guanina/análogos & derivados , Guanina/química , Hipoxantina/química , Mutação , Oxirredução , RecQ Helicases , Especificidade por Substrato , Uracila/química , Helicase da Síndrome de WernerRESUMO
Werner syndrome (WS) is the hallmark premature aging disorder in which affected humans appear older than their chronological age. The protein WRNp, defective in WS, has helicase function, DNA-dependent ATPase, and exonuclease activity. Although WRNp functions in nucleic acid metabolism, there is little or no information about the pathways or protein interactions in which it participates. Here we identify Ku70 and Ku86 as proteins that interact with WRNp. Although Ku proteins had no effect on ATPase or helicase activity, they strongly stimulated specific exonuclease activity. These results suggest that WRNp and the Ku complex participate in a common DNA metabolic pathway.
Assuntos
Antígenos Nucleares , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Cromatografia de Afinidade , DNA Helicases/química , Proteínas de Ligação a DNA/química , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Exodesoxirribonucleases , Exonucleases/metabolismo , Humanos , Autoantígeno Ku , Dados de Sequência Molecular , Proteínas Nucleares/química , Testes de Precipitina , Ligação Proteica , RecQ Helicases , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Helicase da Síndrome de WernerRESUMO
Mutations in the WRN gene result in Werner syndrome, an autosomal recessive disease in which many characteristics of aging are accelerated. A probable role in some aspect of DNA metabolism is suggested by the primary sequence of the WRN gene product. A recombinant His-tagged WRN protein (WRNp) was overproduced in insect cells using the baculovirus system and purified to near homogeneity by several chromatographic steps. This purification scheme removes both nuclease and topoisomerase contaminants that persist following a single Ni(2+)affinity chromatography step and allows for unambiguous interpretation of WRNp enzymatic activities on DNA substrates. Purified WRNp has DNA-dependent ATPase and helicase activities consistent with its homology to the RecQ subfamily of proteins. The protein also binds with higher affinity to single-stranded DNA than to double-stranded DNA. However, WRNp has no higher affinity for various types of DNA damage, including adducts formed during 4NQO treatment, than for undamaged DNA. Our results confirm that WRNp has a role in DNA metabolism, although this role does not appear to be the specific recognition of damage in DNA.
Assuntos
4-Nitroquinolina-1-Óxido/farmacologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Adenosina Trifosfatases/metabolismo , Baculoviridae/genética , DNA Helicases/química , DNA Complementar/análise , Exodesoxirribonucleases , Humanos , Hidrólise , Cinética , RecQ Helicases , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Helicase da Síndrome de WernerRESUMO
Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)-dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40-60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II-dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid-protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , RNA Polimerase II/genética , Transcrição Gênica , Sequência de Aminoácidos , Extratos Celulares , Linhagem Celular , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , Cromatina/genética , DNA Helicases/isolamento & purificação , Exodesoxirribonucleases , Imunofluorescência , Teste de Complementação Genética , Humanos , Dados de Sequência Molecular , Mutação , Plasmídeos/genética , RNA/biossíntese , RecQ Helicases , Sequências Repetitivas de Aminoácidos , Síndrome de Werner/genética , Síndrome de Werner/patologia , Helicase da Síndrome de WernerRESUMO
The human premature aging disorder Werner syndrome (WS) is associated with a large number of symptoms displayed in normal aging. The WRN gene product, a DNA helicase, has been previously shown to unwind short DNA duplexes (=53 base pairs) in a reaction stimulated by single-stranded DNA-binding proteins. We have studied the helicase activity of purified WRN protein on a variety of DNA duplex substrates to characterize the unwinding properties of the enzyme in greater detail. WRN helicase can catalyze unwinding of long duplex DNA substrates up to 849 base pairs in a reaction dependent on human replication protein A (hRPA). Escherichia coli SSB and bacteriophage T4 gene 32 protein (gp32) completely failed to stimulate WRN helicase to unwind long DNA duplexes indicating a specific functional interaction between WRN and hRPA. So far, there have been no reports of any physical interactions between WRN helicase and other proteins. In support of the functional interaction, we demonstrate a direct interaction between WRN and hRPA by coimmunoprecipitation of purified proteins. The physical and functional interaction between WRN and hRPA suggests that the two proteins may function together in vivo in a pathway of DNA metabolism such as replication, recombination, or repair.
Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Compostos Orgânicos , Bacteriófago T4/metabolismo , Benzotiazóis , DNA de Cadeia Simples/metabolismo , Diaminas , Exodesoxirribonucleases , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Peso Molecular , Ligação Proteica , Quinolinas , RecQ Helicases , Proteínas Recombinantes/metabolismo , Proteína de Replicação A , Relação Estrutura-Atividade , Proteínas Virais/metabolismo , Helicase da Síndrome de WernerRESUMO
Heat shock and other stress treatments, resulting in thermotolerance in Neurospora crassa cells, stimulate the induction of a peroxidase at a high level. The putative gene encoding this heat shock-induced peroxidase (hspp) has been cloned, using a cDNA clone of the manganese peroxidase of Phanerochaete chrysosporium, as a probe. Northern blot analysis of total RNA from heat-shocked cell showed the stress-dependent accumulation of a approximately 10 kb transcript. The identity of the HSP, corresponding to the heat-induced peroxidase, was established using hybrid-arrested in vitro translation.