p53 modulates RPA-dependent and RPA-independent WRN helicase activity.
ABSTRACT Werner syndrome is a hereditary disorder characterized by the early onset of age-related symptoms, including cancer. The absence of a p53-WRN helicase interaction may disrupt the signal to direct S-phase cells into apoptosis for programmed cell death and contribute to the pronounced genomic instability and cancer predisposition in Werner syndrome cells. Results from coimmunoprecipitation studies indicate that WRN is associated with replication protein A (RPA) and p53 in vivo before and after treatment with the replication inhibitor hydroxyurea or gamma-irradiation that introduces DNA strand breaks. Analysis of the protein interactions among purified recombinant WRN, RPA, and p53 proteins indicate that all three protein pairs bind with similar affinity in the low nanomolar range. In vitro studies show that p53 inhibits RPA-stimulated WRN helicase activity on an 849-bp M13 partial duplex substrate. p53 also inhibited WRN unwinding of a short (19-bp) forked duplex substrate in the absence of RPA. WRN unwinding of the forked duplex substrate was specific, because helicase inhibition mediated by p53 was retained in the presence of excess competitor DNA and was significantly reduced or absent in helicase reactions catalyzed by a WRN helicase domain fragment lacking the p53 binding site or the human RECQ1 DNA helicase, respectively. p53 effectively inhibited WRN helicase activity on model DNA substrate intermediates of replication/repair, a 5' ssDNA flap structure and a synthetic replication fork. Regulation of WRN helicase activity by p53 is likely to play an important role in genomic integrity surveillance, a vital function in the prevention of tumor progression.
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ABSTRACT: Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects.Nucleic Acids Research 02/2007; 35(22):7527-44. · 8.03 Impact Factor
Article: Comparison of proliferation and genomic instability responses to WRN silencing in hematopoietic HL60 and TK6 cells.[show abstract] [hide abstract]
ABSTRACT: Werner syndrome (WS) results from defects in the RecQ helicase (WRN) and is characterized by premature aging and accelerated tumorigenesis. Contradictorily, WRN deficient human fibroblasts derived from WS patients show a characteristically slower cell proliferation rate, as do primary fibroblasts and human cancer cell lines with WRN depletion. Previous studies reported that WRN silencing in combination with deficiency in other genes led to significantly accelerated cellular proliferation and tumorigenesis. The aim of the present study was to examine the effects of silencing WRN in p53 deficient HL60 and p53 wild-type TK6 hematopoietic cells, in order to further the understanding of WRN-associated tumorigenesis. We found that silencing WRN accelerated the proliferation of HL60 cells and decreased the cell growth rate of TK6 cells. Loss of WRN increased DNA damage in both cell types as measured by COMET assay, but elicited different responses in each cell line. In HL60 cells, but not in TK6 cells, the loss of WRN led to significant increases in levels of phosphorylated RB and numbers of cells progressing from G1 phase to S phase as shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the hyper-activation of homologous recombination repair via up-regulation of RAD51 and BLM protein levels. This resulted in DNA damage disrepair, apparent by the increased frequencies of both spontaneous and chemically induced structural chromosomal aberrations and sister chromatid exchanges. Together, our data suggest that the effects of WRN silencing on cell proliferation and genomic instability are modulated probably by other genetic factors, including p53, which might play a role in the carcinogenesis induced by WRN deficiency.PLoS ONE 01/2011; 6(1):e14546. · 4.09 Impact Factor
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ABSTRACT: It is now clear that large DNA-binding proteins have evolved in mammals to orchestrate the relatively ancient process of DNA recombinational repair. These proteins are recruited to accurately repair DNA double strand breaks (DSBs)--the frequent, potentially lethal and mutagenic lesions in the genomes of all organisms. An essential mammalian regulator of DSB repair is BRCA1. Heterozygous BRCA1 mutations predispose individuals to breast, ovarian and other secondary cancers. BRCA1-defective cells exhibit reduced DSB repair, sensitivity to a wide range of DNA damaging agents, genomic instability and defects in the S-phase checkpoint, transcription and chromatin remodelling. DSBs can be repaired by RAD51/RPA-dependent homologous recombination (HR) or DNA-PK-dependent non-homologous end-joining (NHEJ). Both of these pathways can be imprecise and mutagenic. BRCA1 plays a central role in promoting accurate repair by both HR and NHEJ. Consistent with recent evidence, we have assembled a novel cell-cycle-dependent model in which DNA-PK inhibits RPA in S-phase of the cell cycle, while BRCA1 inhibits the exonuclease processivity of the MRE11/RAD50/NBS1 (MRN) complex and facilitates the removal of RPA in S and G2 phase. This model provides an explanation for how BRCA1 promotes accurate DSB repair during various phases of the cell cycle and also accounts for the dual effects that BRCA1 and MRN activity have upon DNA repair and S-phase arrest.Cell cycle (Georgetown, Tex.) 10/2005; 4(9):1216-22. · 5.36 Impact Factor