Nucleic Acids Research, 2002, Vol. 30 No. 16 3583±3591 Ku heterodimer binds to both ends of the Werner protein and functional interaction occurs at the Werner N-terminus Parimal Karmakar, Carey M. Snowden1, Dale A. Ramsden1 and Vilhelm A. Bohr* Laboratory of Molecular Gerontology, Box 1, National Institute on Aging, IRP, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA and 1Lineberger Comprehensive Cancer Center, Campus Box 7295, Mason Farm Road, Department of Biochemistry and Biophysics and Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599-7295, USA Received April 16, 2002; Revised June 21, 2002; Accepted July 2, 2002 ABSTRACT RecQ, yeast sgs1 and human Bloom (BLM) and Rothmund Thomson (RecQL) proteins. The striking difference between The human Werner syndrome protein, WRN, is a WRN and other members of the RecQ helicase family is the member of the RecQ helicase family and contains N-terminal exonuclease domain of WRN, which shares 3¢®5¢ helicase and 3¢®5¢ exonuclease activities. homology with RNA polymerase I and RNase D of Recently, we showed that the exonuclease activity Escherichia coli (4). of WRN is greatly stimulated by the human Ku Biochemical and genetic evidence suggests that WRN plays heterodimer protein. We have now mapped this an important role in DNA metabolism, possibly by partici- interaction physically and functionally. The Ku70 pating in DNA replication, transcription, repair and/or subunit speci®cally interacts with the N-terminus recombination. Puri®ed recombinant WRN displays both (amino acids 1±368) of WRN, while the Ku80 subunit 3¢®5¢ helicase and 3¢®5¢ exonuclease activity on a variety interacts with its C-terminus (amino acids 940± of DNA substrates (5,6). WS cells are hypersensitive to the 1432). Binding between Ku70 and the N-terminus of DNA-damaging agent 4-nitroquinoline-1-oxide, topoisome- WRN (amino acids 1±368) is suf®cient for stimula- rase inhibitors and DNA interstrand cross-linking agents (4). tion of WRN exonuclease activity. A mutant Ku Thus, WRN is likely to have a role in the DNA damage heterodimer of full-length Ku80 and truncated Ku70 response pathway. This notion is further strengthened by the observation that a number of important cellular proteins that (amino acids 430±542) interacts with C-WRN but not are also involved in DNA damage response pathways with N-WRN and cannot stimulate WRN exonuclease interact with WRN and modulate its catalytic activities. This activity. This emphasizes the functional signi®cance includes human replication protein A (7), p53 (8) and ¯ap of the interaction between the N-terminus of WRN endonuclease 1 (FEN1) (9). and Ku70. The interaction between Ku80 and the We have reported that a factor required for the end joining C-terminus of WRN may modulate some other, as pathway for double-strand break (DSB) repair, the Ku yet unknown, function. The strong interaction heterodimer, interacts with WRN (10,11). WRN exonuclease between Ku and WRN suggests that these two is generally active on the 3¢-recessed strand of a partial DNA proteins function together in one or more pathways duplex. Ku not only stimulates this function, but also relaxes of DNA metabolism. substrate preference, making WRN exonuclease active on substrates like blunt end DNA duplex, 3¢-protruding DNA, single-stranded DNA (12) and DNA containing oxidative INTRODUCTION DNA base lesions (13). Human Werner syndrome (WS) is characterized by early onset In eukaryotic cells, Ku has been implicated as a key of age-associated diseases such as arteriosclerosis, osteo- molecule in DNA DSB repair by the non-homologous end porosis and diabetes mellitus type II (1). Moreover, the joining (NHEJ) pathway (14). Ku binds to the broken DNA patients display high levels of genomic instability and are ends and recruits several other factors to DNA ends that are prone to cancer (2). In culture, WS cells exhibit replicative required for ef®cient NHEJ, including DNA-PKcs (the senescence, extended S phase and a variety of chromosomal catalytic subunit of DNA-activated protein kinase) and the aberrations, including translocations, insertions, deletions, etc. XRCC4±ligase IV complex (15,16). (1). The Werner gene (wrn) encodes a protein of 1432 amino NHEJ often involves signi®cant processing of broken ends acids (WRN) (3). It is a member of the RecQ family of before joining can occur, but the identity of the processing helicases. Seven conserved helicase motifs in the central factors are still only partly known. The strong physical and region of the WRN polypeptide share homology with bacterial functional interaction between WRN and Ku suggests that the *To whom correspondence should be addressed. Tel: +1 410 558 8223; Fax: +1 410 558 8157; Email: [email protected] 3584 Nucleic Acids Research, 2002, Vol. 30 No. 16 exonuclease activity of WRN might participate in the from Santa Cruz Biotechnology. Mouse monoclonal anti-poly- processing of DNA ends during NHEJ. Cells de®cient in histidine antibody was obtained from Sigma Chemical Co. WRN, Ku70 or Ku80 all show genomic instability and undergo premature replicative senescence (17), consistent Far western analysis with the suggestion that WRN and Ku act in a common Far western blotting analysis was conducted essentially as pathway in DNA metabolism (13). described elsewhere (20). Brie¯y, 0.2±1.0 mg each poly- Recently, another laboratory reported that the N-terminus of peptide was electrophoresed on 8±16% acrylamide WRN interacts with amino acids 215±276 of Ku80 (11,12). Tris±glycine gels and transferred to PVDF membranes However, that study utilized in vitro translated Ku and (Amersham). All subsequent steps were performed at 4°C. included no analysis of the WRN±Ku functional interaction to Membranes were immersed twice in denaturation buffer (6 M substantiate the physical interaction. We undertook the current studies to map the region(s) of interaction between WRN and guanidine±HCl in PBS) for 10 min followed by 6 3 10 min Ku. We report here, using several approaches, that both the N- washes in serial dilutions (1:1) of denaturation buffer in PBS and C-termini of WRN can interact independently with Ku. supplemented with 1 mM DTT. Membranes were blocked in The C-terminus of WRN interacts with the Ku80 subunit, TBS containing 10% powdered milk, 0.3% Tween-20 for while the N-terminus of WRN interacts with the Ku70 subunit. 30 min before being incubated with puri®ed proteins in TBS We further show that the interaction between WRN and Ku80 supplemented with 0.25% powdered milk, 0.3% Tween-20, is not required for stimulation of exonuclease activity. 1 mM DTT and 1 mM PMSF for 60 min. Membranes were then washed for 4 3 10 min in TBS containing 0.3% Tween-20, 0.25% powdered milk. The second wash contained MATERIALS AND METHODS 0.0001% glutaraldehyde. Conventional western analysis was then performed to detect the presence of interacting proteins. Proteins Appropriate horseradish peroxidase-conjugated secondary Baculovirus constructs for recombinant hexa-histidine tagged antibody (Vector Laboratories) was used and the signal was full-length WRN protein or a truncated version of WRN detected using ECL (Amersham) following the manufacturer's (N-terminal 368 amino acids, designated N-WRN) were instructions. kindly provided by Dr Matthew Gray (University of Washington, Seattle, WA). Ampli®ed baculovirus was used Pull-down assay to infect sf9 insect cells for overexpression of WRN protein as Insect cells infected with baculovirus containing Ku mutant or previously described (18). The protein was puri®ed by passing wild-type Ku were lysed in 150 mM NaCl, 10 mM Tris pH 7.4, through DEAE±Sepharose (Pharmacia), Q Sepharose 1% Triton X-100, 0.2 mM PMSF, 20 mg/ml aprotinin, (Pharmacia) and Ni±NTA (Gibco BRL) chromatography 10 mg/ml leupeptin, 10 U/ml DNase. The suspension was (18). Purity of the protein was routinely checked by then centrifuged at 12 000 r.p.m. in a Beckman Avanti J-25I Coomassie staining. A recombinant hexa-histidine tagged centrifuge for 20 min at 4°C. The supernatant was incubated C-terminal fragment of WRN (residues 940±1432, designated with ~100 ng puri®ed proteins (either WRN or its C- or C-WRN) was overexpressed in E.coli and puri®ed as previ- N-terminal fragments) for 1 h at 4°C followed by incubation ously described (10). Human Ku heterodimer was over- with appropriate antibody for 4 h at 4°C. The immune expressed and puri®ed using baculoviral constructs as complex was then precipitated with protein G plus agarose previously reported (16). A Ku80 truncation mutant (1±571) (Santa Cruz Biotechnology) and washed three times with was made by PCR amplifcation of the full-length human 150 mM NaCl, 10 mM Tris pH 7.4, 1% Triton X-100, 10 U/ml cDNA using the primers 80N (5¢-acaggaattcatggtgcggtcggg- DNase. Immunoprecipitated proteins were then incubated gaataaggca) and 80571STOP (5¢-agtctagactactcagtctttaattttt- with SDS loading buffer at 90°C for 5 min and separated in tagctgtaggt), followed by insertion into pFASTBAC1 (Life an 8±16% polyacrylamide Tris±glycine gel. Conventional Technologies). Virus stocks expressing truncation mutants western blotting was performed to identify the proteins. Ku70(330±609) and Ku70(430±542) (19) were the gift of Dr Wesley Reeves (University of Florida). Exonuclease substrates Materials Single-stranded DNA oligomers (72mer and 53mer or 32mer [g-32P]ATP was purchased from New England Nuclear. ATP and 43mer) were obtained from Gibco BRL. The 53mer or was purchased from Amersham Pharmacia Biotech. 32mer (7 pmol) was 5¢-labeled with [g-32P]ATP (60 mCi, 3000 mCi/mmol) and polynucleotide kinase (10 U) under Antibodies standard conditions.