Format

Send to

Choose Destination
Methods Enzymol. 2006;409:52-85.

Enzymatic mechanism of the WRN helicase/nuclease.

Author information

1
Laboratory of Molecular Gerontology, National Institute of Aging-IRP, National Institutes of Health, Baltimore, Maryland, USA.

Abstract

Werner syndrome (WS) is a premature aging disorder characterized by genomic instability and increased cancer risk (Martin, 1978). The WRN gene product defective in WS belongs to the RecQ family of DNA helicases (Yu et al., 1996). Mutations in RecQ family members BLM and RecQ4 result in two other disorders associated with elevated chromosomal instability and cancer, Bloom syndrome and Rothmund-Thomson syndrome, respectively (for review see Opresko et al., 2004a). RecQ helicase mutants display defects in DNA replication, recombination, and repair, suggesting a role for RecQ helicases in maintaining genomic integrity. The WRN gene encodes a 1,432 amino acid protein that has several catalytic activities (Brosh and Bohr, 2002) (Fig. 1). WRN is a DNA-dependent ATPase and utilizes the energy from ATP hydrolysis to unwind double-stranded DNA. WRN is also a 3' to 5' exonuclease, consistent with the presence of three conserved exonuclease motifs homologous to the exonuclease domain of Escherichia coli DNA polymerase I and RNase D. Most recently, WRN (Machwe et al., 2005) and other human RecQ helicases (Garcia et al., 2004; Machwe et al., 2005; Sharma et al., 2005) have been reported to possess an intrinsic single-strand annealing activity. In addition to its catalytic activities, WRN interacts with a number of proteins involved in various aspects of DNA metabolism. To understand the role of WRN in the maintenance of genome stability, a number of laboratories have undertaken a thorough characterization of its molecular and cellular functions. Here, we describe methods and approaches used for the functional and mechanistic analysis of WRN helicase or exonuclease activity. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding or exonuclease activity of WRN protein are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of WRN or related helicases or nucleases, which would serve as a platform for further investigation of its molecular and cellular functions.

PMID:
16793395
DOI:
10.1016/S0076-6879(05)09004-X
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center