Identification of a Coiled Coil in Werner Syndrome Protein That Facilitates Multimerization and Promotes Exonuclease Processivity

J Jefferson P Perry,Aroumougame Asaithamby,Adam Barnebey,Foad Kiamanesch,David J Chen,Seungil Han,John A Tainer,Steven M Yannone

doi:10.1074/jbc.m110.124941

Abstract

Werner syndrome (WS) is a rare progeroid disorder characterized by genomic instability, increased cancer incidence, and early onset of a variety of aging pathologies. WS is unique among early aging syndromes in that affected individuals are developmentally normal, and phenotypic onset is in early adulthood. The protein defective in WS (WRN) is a member of the large RecQ family of helicases but is unique among this family in having an exonuclease. RecQ helicases form multimers, but the mechanism and consequence of multimerization remain incompletely defined. Here, we identify a novel heptad repeat coiled coil region between the WRN nuclease and helicase domains that facilitates multimerization of WRN. We mapped a novel and unique DNA-dependent protein kinase phosphorylation site proximal to the WRN multimerization region. However, phosphorylation at this site affected neither exonuclease activity nor multimeric state. We found that WRN nuclease is stimulated by DNA-dependent protein kinase independently of kinase activity or WRN nuclease multimeric status. In addition, WRN nuclease multimerization significantly increased nuclease processivity. We found that the novel WRN coiled coil domain is necessary for multimerization of the nuclease domain and sufficient to multimerize with full-length WRN in human cells. Importantly, correct homomultimerization is required for WRN function in vivo as overexpression of this multimerization domain caused increased sensitivity to camptothecin and 4-nitroquinoline 1-oxide similar to that in cells lacking functional WRN protein.

Highlights

Werner syndrome (WS) is a rare progeroid disorder characterized by genomic instability, increased cancer incidence, and early onset of a variety of aging pathologies
We found that which encodes a 160-kDa protein (WRN) nuclease is stimulated by DNA-dependent protein kinase independently of kinase activity or WRN nuclease multimeric status
Members of the RecQ family are reported to exist in different multimeric forms, and the relationships between multimerization states and functionality remain an active area of research

Summary

That Facilitates Multimerization and Promotes Exonuclease Processivity*

We mapped a novel and unique DNA-dependent protein kinase phosphorylation site proximal to the WRN multimerization region. Phosphorylation at this site affected neither exonuclease activity nor multimeric state. WS arises due to hereditary defects in the WRN gene, which encodes a 160-kDa protein (WRN) containing a central 3Ј–5Ј DNA helicase domain that has sequence homology to the Escherichia coli RecQ helicase, whose roles in DNA metabolism remain incompletely defined (8 –10). The pathologies of the three RecQ-associated diseases appear to be quite distinct, suggesting that WRN, BLM, and RecQL4 have discrete functions within the cell. WRN-exo has gained an additional function over the DNA polymerase I proofreading exonuclease as WRN-exo displays increased enzymatic activity in the presence of the Ku70/80 subunit of DNA-dependent protein kinase (DNA-PK) [14].

WRN Multimerization Domain

EXPERIMENTAL PROCEDURES

RESULTS

Dynamic light scattering measurements for three WRN nuclease constructs

WRN Molecular weight Population Radius Calculated mass aa

DISCUSSION