DNA Repair 5 (2006) 172–180 Biochemical characterization of the RECQ4 protein, mutated in Rothmund-Thomson syndrome Margaret A. Macris a , Lumir Krejci a,∗,1 , Wendy Bussen b , Akira Shimamoto c , Patrick Sung a,∗ a Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar St., C130 Sterling Hall of Medicine, New Haven, CT 06520-8024, USA b Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio, TX 78245-3207, USA c Department of Target Discovery, GeneCare Research Institute, 200 Kajiwara, Kamakura, Kanagawa 247-0063, Japan Received 24 June 2005; received in revised form 31 August 2005; accepted 2 September 2005 Available online 7 October 2005 Abstract Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by growth deficiency, skin and skeletal abnormalities, and a predisposition to cancer. Mutations in the RECQ4 gene, one of five human homologs of the E. coli recQ gene, have been identified in a subset of RTS patients. Cells derived from RTS patients show high levels of chromosomal instability, implicating this protein in the maintenance of genomic integrity. However, RECQ4 is the least characterized of the RecQ helicase family with regard to its molecular and catalytic properties. We have expressed the human RECQ4 protein in E. coli and purified it to near homogeneity. We show that RECQ4 has an ATPase function that is activated by DNA, with ssDNA being much more effective than dsDNA in this regard. We have determined that a DNA length of 60 nucleotides is required to maximally activate ATP hydrolysis by RECQ4, while the minimal site size for ssDNA binding by RECQ4 is between 20 and 40 nucleotides. Interestingly, RECQ4 possesses a single-strand DNA annealing activity that is inhibited by the single-strand DNA binding protein RPA. Unlike the previously characterized members of the RecQ family, RECQ4 lacks a detectable DNA helicase activity. © 2005 Elsevier B.V. All rights reserved. Keywords: RTS; RECQ4; ATPase; Helicase; ssDNA annealing; DNA repair 1. Introduction DNA helicases play essential roles in DNA metabolism, including replication, transcription, recombination, and repair. The RecQ helicase family has been implicated in double-strand break repair and homologous recombination and is important for the maintenance of genomic integrity [1]. Members of this protein family have been identified from E. coli to humans [1]. While yeast and bacteria possess a single RecQ helicase, there are five apparent orthologs in humans. Mutations in three human RecQ-like helicases, BLM (RECQ2), WRN (RECQ3), and RECQ4 have been associated with Bloom’s syndrome (BS), Werner’s syndrome (WS), and Rothmund-Thomson syndrome ∗ Corresponding authors. Tel.: +1 203 785 4553; fax: +1 203 785 6037. E-mail addresses: lumir.krejci@yale.edu (L. Krejci), patrick.sung@yale.edu (P. Sung). 1 Note: As of August 1, 2005, Dr. Lumir Krejci can be contacted at National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, Brno 611 37, Czech Republic. (RTS), respectively. Patients with all three of these disorders show increased cancer susceptibility and genomic instability [2,3], although the clinical features of the disorders and spectrum of cancers observed in the patients are different. RTS is an autosomal recessive disorder characterized by growth deficiency, juvenile cataracts, skin hyperpigmentation, poikiloderma, brittle hair, photosensitivity, congenital skeletal defects, and a predisposition to malignancy, especially osteosar- comas [4–6]. Over half of patients with RTS harbor mutations in the RECQ4 gene [7–11]. The RECQ4 gene encodes a 1208 amino acid protein with a predicted molecular weight of 133 kDa [12,13] and contains sequence features that define the RecQ fam- ily [1]. Most of the identified mutations in the RTS patients are within the conserved helicase domain of RECQ4 spanning exons 8–14 [7–9], and these mutations are predicted to produce pro- teins truncated in this domain. Aside from the helicase domain, RECQ4 lacks significant homology to other known proteins. The RECQ4 protein is normally present in the nucleus, but in certain transformed cells, it becomes overexpressed and a majority of it is found in the cytoplasm, where it seems to associate with the 1568-7864/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2005.09.005