Article

p53 modulates RPA-dependent and RPA-independent WRN helicase activity.

Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, Maryland, USA.
Cancer Research (impact factor: 7.86). 03/2005; 65(4):1223-33. DOI:10.1158/0008-5472.CAN-03-0231 pp.1223-33
Source: PubMed

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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Keywords

5' ssDNA flap structure
 
849-bp M13 partial duplex substrate
 
cancer predisposition
 
direct S-phase cells
 
excess competitor DNA
 
forked duplex substrate
 
genomic integrity surveillance
 
helicase reactions catalyzed
 
human RECQ1 DNA helicase
 
low nanomolar range
 
model DNA substrate intermediates
 
p53 binding site
 
p53-WRN helicase interaction
 
pronounced genomic instability
 
purified recombinant WRN
 
replication protein
 
three protein pairs bind
 
tumor progression
 
Werner syndrome cells
 
WRN helicase domain fragment