Junction of RecQ Helicase Biochemistry and Human Disease

RecQ helicases are a family of conserved enzymes required for maintaining the genomic integrity, that function as suppressors of inappropriate recombination. Mutations in Escherichia coli RecQ and in the Saccharomyces cerevisiae RecQ homolog, Sgs1, result in an increased frequency of illegitimate recombination (1Bachrati C.Z. Hickson I.D. Biochem. J. 2003; 374: 577-606Google Scholar). In humans, defects in three RecQ family proteins are associated with rare autosomal-recessive disorders characterized by genomic instability and increased cancer susceptibility. Mutations in WRN, BLM, and RECQ4 give rise to the disorders Werner syndrome (WS), 1The abbreviations used are: WS, Werner syndrome; BS, Bloom syndrome; RTS, Rothmund-Thomson syndrome; ss, single strand; HJ, Holliday junction; HR, homologous recombination; NHEJ, non-homologous end joining; DSB, double strand break; SSB, single strand break; RPA, replication protein A; BER, base excision repair; ALT, alternative lengthening of telomeres; RQC, RecQ C-terminal region; HRDC, helicase RNase D C-terminal domain; PK, protein kinase. Bloom syndrome (BS), and Rothmund-Thomson syndrome (RTS), respectively, the clinical features of which have been reviewed elsewhere (2Hickson I.D. Nat. Rev. Cancer. 2003; 3: 169-178Google Scholar). Briefly, BS patients are predisposed to many types of cancer with the mean age of onset of 24. WS patients are especially predisposed to sarcomas, premature aging, and age-associated diseases. RTS patients have a characteristic rash, poikiloderma, and are predisposed to osteosarcomas and some features of premature aging. The molecular basis of genomic instability and premature aging is not well understood. The RecQ family is named after E. coli RecQ helicase, a well characterized prototypical member (Fig. 1). Helicases separate complementary strands of nucleic acids in a reaction coupled to NTP hydrolysis. RecQ helicases have a common helicase domain, which binds and hydrolyzes ATP. Most RecQ helicases have a highly conserved multifunctional RecQ C-terminal region (RQC) and a helicase RNase D C-terminal (HRDC) domain (Fig. 1). A recent report of the x-ray crystal structure for the E. coli RecQ catalytic core indicates that the RQC domain contains DNA and protein binding motifs (3Bernstein D.A. Zittel M.C. Keck J.L. EMBO J. 2003; 22: 4910-4921Google Scholar). Consistent with this, the RQC domain of WRN binds to various DNA substrates and mediates interactions with other proteins involved in DNA metabolism (4von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Google Scholar). The E. coli RecQ HRDC domain is required for stable DNA binding but not for catalytic activity (5Bernstein D.A. Keck J.L. Nucleic Acids Res. 2003; 31: 2778-2785Google Scholar). Similarly, the HRDC domain of human WRN also binds DNA substrates but is not required for catalytic activity (4von Kobbe C. Thoma N.H. Czyzewski B.K. Pavletich N.P. Bohr V.A. J. Biol. Chem. 2003; 278: 52997-53006Google Scholar). Two RecQ family proteins WRN and Xenopus laevis FFA-1 also have an exonuclease domain. Bacteria and yeast have a single RecQ family member, and up to five RecQ members have been found in mammals. To better define the precise roles of RecQ helicases in vivo, significant research effort has been devoted to characterizing the biochemical properties of RecQ helicases and to identifying important protein interactions between RecQ helicases and other well characterized proteins. This review will focus primarily on these aspects and on the most well characterized RecQ helicases, due to space limitations. RecQ helicase genetic studies in yeast have been recently reviewed elsewhere (6Khakhar R.R. Cobb J.A. Bjergbaek L. Hickson I.D. Gasser S.M. Trends Cell Biol. 2003; 13: 493-501Google Scholar). All RecQ helicases purified and characterized to date unwind duplex DNA in a 3′ to 5′ direction with respect to the DNA strand bound by the helicase. Substrate preferences are determined by comparing product amounts, reaction kinetics, and/or protein affinities for the substrates in side-by-side reactions. E. coli RecQ has broad DNA substrate specificity and acts on DNA duplexes containing blunt or forked termini, duplexes with 3′ or 5′ single strand (ss) tails, D-loops, and 3- or 4-way (Holliday) junctions (7Harmon F.G. Kowalczykowski S.C. Genes Dev. 1998; 12: 1134-1144Google Scholar) (Fig. 2). Mammalian and yeast RecQ helicases are less promiscuous than E. coli RecQ, which may reflect the DNA binding specificities of their unique N- and C-terminal protein domains. S. cerevisiae Sgs1 and human BLM and WRN do not unwind duplexes with blunt termini or with 5′ ssDNA tails (Fig. 2), but these enzymes preferentially unwind forked duplexes with branched structures or junctions (8Bennett R.J. Keck J.L. Wang J.C. J. Mol. Biol. 1999; 289: 235-248Google Scholar, 9Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Scopus (486) Google Scholar). WRN and BLM recognize and specifically bind to junction sites and have higher relative affinity for substrates with junctions (10Shen J.C. Loeb L.A. Nucleic Acids Res. 2000; 28: 3260-3268Google Scholar, 11Orren D.K. Theodore S. Machwe A. Biochemistry. 2002; 41: 13483-13488Google Scholar, 12van Brabant A.J. Ye T. Sanz M. German III, J.L. Ellis N.A. Holloman W.K. Biochemistry. 2000; 39: 14617-14625Google Scholar, 13Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Google Scholar). They also preferentially unwind a Holliday junction (HJ) substrate with short arms constructed from oligonucleotides, compared with a forked duplex (9Mohaghegh P. Karow J.K. Brosh Jr., J.R. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Scopus (486) Google Scholar), and promote extensive branch migration (several kilobases) of long-arm HJ substrates generated by RecA protein (α-structure) (2Hickson I.D. Nat. Rev. Cancer. 2003; 3