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. 2010 Aug 13;285(33):25699-707.
doi: 10.1074/jbc.M110.124941. Epub 2010 Jun 1.

Identification of a coiled coil in werner syndrome protein that facilitates multimerization and promotes exonuclease processivity

J Jefferson P Perry  1 , Aroumougame Asaithamby, Adam Barnebey, Foad Kiamanesch, David J Chen, Seungil Han, John A Tainer, Steven M Yannone
Affiliations

Identification of a coiled coil in werner syndrome protein that facilitates multimerization and promotes exonuclease processivity

J Jefferson P Perry et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Limited proteolysis of WRN exonuclease defines domain boundaries. A, Coomassie-stained 12% SDS-PAGE gel of limited proteolysis of WRN(1–333) purified from E. coli with either chymotrypsin (C) or trypsin (T) and incubated for the noted times. B, three purified recombinant WRN nuclease constructs were produced and are shown (gel inset); construct size and position relative to full-length WRN and the RecQ and Helicase and RNase-D C-terminal (HRDC) domains are schematically illustrated.
FIGURE 2.
FIGURE 2.
WRN(1–333) is trimeric whereas smaller nuclease constructs are primarily monomeric. A, overlay of three separate S-200 gel filtration elution profiles for WRN(1–333) (red), WRN(1–236) (blue), and WRN(38–236) (black) in buffer containing 0.5 m NaCl. B, standard curve based on ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa), and chymotrypsinogen A (25 kDa). The elution volume intercepts for three WRN constructs are indicated by color-coded arrows.
FIGURE 3.
FIGURE 3.
WRN multimerization domain forms highly stable multimers and contains a coiled coil motif. A, multiple sequence alignment of Homo sapiens WRN amino acids 247–299 with Pan troglodyte, Equus caballus, Canis familiaris, Bos taurus, Mus musculus, Rattus norvegicus, Monodelphis domestica, Ornithorhynchus anatinus, Gallus gallus, Xenopus laevis, Xenopus tropicalis, and Taeniopygia guttata WRN homologues reveals a conserved heptad repeat coiled coil motif. Heptad positions 1 and 4 are highlighted in blue and green, respectively; protein species names and start and end amino acids are denoted. Asterisks denote a conserved break in the heptad pattern of WRN proteins. Human RecQ1 coiled coil is shown for comparison (bottom). B, SDS-PAGE of recombinant His6-WRN(221–333) nickel resin pull-down experiments; extract (Input), nickel bead supernatant (Sup.) and nickel resin eluate (Eluate) are indicated. C, Western blot of the same experiment using rabbit polyclonal antibodies recognizing the epitope(s) within WRN(236–333). D, Western blot of repeated experiment, including E. coli control lane probed with antibodies recognizing polyhistidine.
FIGURE 4.
FIGURE 4.
WRN coiled coil domain multimerizes with WRN in vivo and alters WRN function. A, immunoprecipitations (IP) of extracts from HeLa cells overexpressing FLAG-WRN(250–366); antibodies used for immunoprecipitation (above) and proteins detected are indicated (left), full length WRN (WRN-FL). B–D, colony formation assays comparing the survival of parental HeLa cells (solid black circles) with the survival of HeLa cells expressing FLAG-WRN(250–366) (open circles) to the indicated doses of camptothecin (B), 4-nitroquinoline 1-oxide (C), and hydroxyurea (D). All assays were carried out in triplicate; the average values are plotted, and the standard deviation (error bars) is indicated.
FIGURE 5.
FIGURE 5.
DNA-PK phosphorylates WRN-exo specifically at Ser-319 within the multimerization region. A, in vitro kinase assays containing ∼500 ng of DNA-PK and [32P]ATP resolved by SDS-PAGE. Lane 1, DNA-PK reaction alone; lane 2, DNA-PK reaction with ∼50 ng of full-length WRN; lane 3, DNA-PK reaction with ∼1 μg of WRN(1–333); lane 4, DNA-PK with ∼1.5 μg of WRN(38–236). The Coomassie Blue-stained gel (left panel) and the phosphorimage of the same gel (right panel) are shown. Reaction component proteins are indicated (left). B, DNA-PK phosphorylates the WRN nuclease domain specifically at serine 319. Partially purified WRN(1–333), either wild type (WT) Ser-319, S319A mutation, or S323A mutation, was incubated and resolved on SDS-PAGE as above, stained with Coomassie Blue, and exposed to a phosphorimage screen (top panel) or photographed (middle panel), or a parallel gel was transferred to nitrocellulose and probed with an antibody recognizing the nuclease domain of WRN (bottom panel). The proximal amino acid sequence is shown with Ser-319 (large red) and Ser-323 (small red) indicated.
FIGURE 6.
FIGURE 6.
DNA-PK phosphorylation of WRN(1–333) does not alter multimeric state or nuclease activity. A, overlaid elution profiles from S-200 gel filtration of saturating kinase reactions (red) and phosphatase reactions (black) containing equivalent amounts of WRN(1–333). WRN(1–333) and phosphatase (PPase) protein and ATP peaks are indicated. B, nuclease assays with double-stranded DNA substrates with stoichiometrically equivalent amounts of the indicated WRN constructs supplemented with the noted components incubated at 37 °C for 30 min and resolved by urea-PAGE (S indicates a mock reaction containing no added protein).
FIGURE 7.
FIGURE 7.
WRN exonuclease multimeric state influences enzymatic activity. A, exonuclease assay of increasing molar amounts of WRN(1–333), WRN(1–236), and WRN(38–236) from left to right as indicated by the triangles (100, 300, and 900 fmol). Reactions were incubated for 30 min and resolved by urea-PAGE; major points of exonuclease termination are noted as “P1” and “P2.” B, schematic illustration of the relative position of the core nuclease domain, the coiled coil multimerization domain, and the DNA-PK phosphorylation site. C, homology model of hexameric RecQ helicase and WRN exonuclease bridged by two three-stranded coiled coils, showing a possible dimer of trimers WRN architecture. The carboxyl-terminal tail of the WRN exonuclease and amino termini of RecQ helicase structures guided placement and orientation of the 50-amino acid coiled coil structures.
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