Abstract
Werner protein (WRN), member of the RecQ helicase family, is a helicase and exonuclease, and participates in multiple DNA metabolic processes including DNA replication, recombination and DNA repair. Mutations in the WRN gene cause Werner syndrome, associated with premature aging, genome instability and cancer predisposition. The RecQ C-terminal (RQC) domain of WRN, containing α2-α3 loop and β-wing motifs, is important for DNA binding and for many protein interactions. To better understand the critical functions of this domain, we generated recombinant WRN proteins (using a novel purification scheme) with mutations in Arg-993 within the α2-α3 loop of the RQC domain and in Phe-1037 of the -wing motif. We then studied the catalytic activities and DNA binding of these mutant proteins as well as some important functional protein interactions. The mutant proteins were defective in DNA binding and helicase activity, and interestingly, they had deficient exonuclease activity and strand annealing function. The RQC domain of WRN has not previously been implicated in exonuclease or annealing activities. The mutant proteins could not stimulate NEIL1 incision activity as did the wild type. Thus, the Arg-993 and Phe-1037 in the RQC domain play essential roles in catalytic activity, and in functional interactions mediated by WRN.
Highlights
Werner Syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging
The FLAG tag was not employed during purification, and was placed at the C-terminus of Werner protein (WRN) to be utilized in future experiments
A recent crystallographic study suggested that the winged-helix motif of the RecQ C-terminal (RQC) domain could play a central role in the DNA unwinding activity of WRN [8]
Summary
Werner Syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. Cells from WS patients show elevated levels of DNA deletions, translocations, chromosomal breaks, and display replicative defects, including an extended S-phase and premature senescence [1, 2]. The WRN gene, encoding Werner protein, has been identified as defective in WS. WRN belongs to the RecQ helicase family, members of which are ubiquitously conserved from bacteria to human [3], and have been implicated in various DNA metabolic pathways, including DNA replication, recombination, and DNA repair [4, 5]. WRN exhibits DNA-dependent ATPase, ATP dependent 3’→5’ DNA helicase, single strand DNA annealing and exonuclease activities. WRN protein consists of 1,432 amino acid residues with multiple domains, including helicase (ATPase), exonuclease, RecQ C-terminal (RQC), and helicase-and-RNaseD-like-C-terminal (HRDC) domains (Figure 1A). The domain is important for protein function, since its deletion resulted in significant decrease in helicase activity of WRN [6] and BLM [7], and since most of the WRN protein partners interact with either entire, or at least with a part of this region [5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
