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

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.

Multimerization domains are associated with apparent strand exchange activity in BLM and WRN DNA helicases

“…1A). Most of this sequence (i.e., R235 - D526) is poorly conserved among WRN orthologs, however this region was recently shown to contain a conserved 53 aa heptad-repeat coiled-coil motif (L247-I299) that promotes WRN multimerization [11]. We therefore created chimeric Sgs1 proteins in which the Sgs1-SE domain was replaced by WRN 235–526 or two N-terminal truncations of this domain, WRN 255–526 and WRN 301–526 .…”

“…The WRN protein is unique among the RecQ family members in that it contains a 3’–5’ exonuclease domain in its N-terminus [6–10] and the N-terminus contains a coiled-coil domain that is important for proper multimerization in vivo [11] (Fig. 1A).…”

RECQL1 and WRN Proteins Are Potential Therapeutic Targets in Head and Neck Squamous Cell Carcinoma

“…The epigenetic inactivation of WRN leads to repress WRN proteins, and increases chromosomal instability and sensitivity to chemotherapeutic drugs in cultured cells; and enhances the clinical sensitivity to camptothecin in human colorectal tumors (22). WRN interacting with DNA-PKcs and/or KU70-80 may participate in DNA repair of double-strand breaks through the nonhomologous end-joining pathway (39)(40)(41)(42). WRN also participates in telomere maintenance (11,18).…”

Cooperative DNA Binding and Protein/DNA Fiber Formation Increases the Activity of the Dnmt3a DNA Methyltransferase

“…For a DNA methyltransferase this means that the enzyme could subsequently methylate several cytosines on one DNA molecule without ever leaving the DNA because it can move along the DNA after each turnover to reach the next target site. Processivity has been shown for many DNA enzymes like DNA polymerases (15), RNA polymerases (16), nucleases (17,18), restriction enzymes (19), and also Dnmts like M.SssI (20), EcoDam (21), or Dnmt1 (22)(23)(24). Processive DNA methylation has also been reported for Dnmt3a by the HolzSchietinger and Reich (25).…”