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• W2010606793 abstract "Helicases are among the first enzymes to encounter DNA damage during DNA processing within the cell and thus are likely to be targets for the adverse effects of DNA lesions induced by environmental chemicals. Here we examined the effect of cis- and trans-opened 3,4-diol 1,2-epoxide (DE) DNA adducts of benzo[c]phenanthrene (BcPh) at N 6 of adenine on helicase activity. These adducts are derived from the highly tumorigenic (–)-(1R,2S,3S,4R)-DE as well as its less carcinogenic (+)-(1S,2R,3R,4S)-DE enantiomer in both of which the benzylic 4-hydroxyl group and epoxide oxygen are trans. The hydrocarbon portions of these adducts intercalate into DNA on the 3′ or the 5′ side of the adducted deoxyadenosine for the 1S- and 1R-adducts, respectively. These adducts inhibited the human Werner (WRN) syndrome helicase activity in a strand-specific and stereospecific manner. In the strand along which WRN translocates, cis-opened adducts were significantly more effective inhibitors than trans-opened isomers, indicating that WRN unwinding is sensitive to adduct stereochemistry. WRN helicase activity was also inhibited but to a lesser extent by cis-opened BcPh DE adducts in the displaced strand independent of their direction of intercalation, whereas inhibition by the trans-opened stereoisomers in the displaced strand depended on their orientation, such that only adducts oriented toward the advancing helicase inhibited WRN activity. A BcPh DE adduct positioned in the helicase-translocating strand did not sequester WRN, nor affect the rate of ATP hydrolysis relative to an unadducted control. Although the Bloom (BLM) syndrome helicase was also inhibited by a cis-opened adduct in a strand-specific manner, this helicase was not as severely affected as WRN. Because BcPh DEs form substantial amounts of deoxyadenosine adducts at dA, their adverse effects on helicases could contribute to genetic damage and cell transformation induced by these DEs. Thus, the unwinding activity of RecQ helicases is sensitive to the strand, orientation, and stereochemistry of intercalated polycyclic aromatic hydrocarbon adducts. Helicases are among the first enzymes to encounter DNA damage during DNA processing within the cell and thus are likely to be targets for the adverse effects of DNA lesions induced by environmental chemicals. Here we examined the effect of cis- and trans-opened 3,4-diol 1,2-epoxide (DE) DNA adducts of benzo[c]phenanthrene (BcPh) at N 6 of adenine on helicase activity. These adducts are derived from the highly tumorigenic (–)-(1R,2S,3S,4R)-DE as well as its less carcinogenic (+)-(1S,2R,3R,4S)-DE enantiomer in both of which the benzylic 4-hydroxyl group and epoxide oxygen are trans. The hydrocarbon portions of these adducts intercalate into DNA on the 3′ or the 5′ side of the adducted deoxyadenosine for the 1S- and 1R-adducts, respectively. These adducts inhibited the human Werner (WRN) syndrome helicase activity in a strand-specific and stereospecific manner. In the strand along which WRN translocates, cis-opened adducts were significantly more effective inhibitors than trans-opened isomers, indicating that WRN unwinding is sensitive to adduct stereochemistry. WRN helicase activity was also inhibited but to a lesser extent by cis-opened BcPh DE adducts in the displaced strand independent of their direction of intercalation, whereas inhibition by the trans-opened stereoisomers in the displaced strand depended on their orientation, such that only adducts oriented toward the advancing helicase inhibited WRN activity. A BcPh DE adduct positioned in the helicase-translocating strand did not sequester WRN, nor affect the rate of ATP hydrolysis relative to an unadducted control. Although the Bloom (BLM) syndrome helicase was also inhibited by a cis-opened adduct in a strand-specific manner, this helicase was not as severely affected as WRN. Because BcPh DEs form substantial amounts of deoxyadenosine adducts at dA, their adverse effects on helicases could contribute to genetic damage and cell transformation induced by these DEs. Thus, the unwinding activity of RecQ helicases is sensitive to the strand, orientation, and stereochemistry of intercalated polycyclic aromatic hydrocarbon adducts. Helicases are enzymes that disrupt complementary strands of duplex DNA in a reaction dependent on nucleoside-5′-triphosphate hydrolysis (1Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, 2Marians K.J. Struct. Fold. Des. 2000; 8: R227-R235Abstract Full Text Full Text PDF Scopus (34) Google Scholar, 3Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (459) Google Scholar). Evidence suggests that helicases play important roles in DNA metabolism, and a growing number of helicases have been linked to human disease (4Mohaghegh P. Hickson I.D. Hum. Mol. Genet. 2001; 10: 741-746Crossref PubMed Scopus (188) Google Scholar, 5van Brabant A.J. Stan R. Ellis N.A. Annu. Rev. Genomics Hum. Genet. 2000; 1: 409-459Crossref PubMed Scopus (205) Google Scholar). Pathways of DNA replication, recombination, and repair are affected by DNA lesions, and the effects of helicases on such pathways are probably modulated by their interactions with chemically modified DNA. Although the mechanism for DNA unwinding has been studied for several helicases (reviewed in Refs. 1Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (669) Google Scholar, 2Marians K.J. Struct. Fold. Des. 2000; 8: R227-R235Abstract Full Text Full Text PDF Scopus (34) Google Scholar, 3Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (459) Google Scholar), very little is known regarding how specific covalently linked adducts affect helicase function.RecQ helicases are believed to function in repairing replication forks that have been stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur (6Wu L. Hickson I.D. Mutat. Res. 2002; 509: 35-47Crossref PubMed Scopus (36) Google Scholar). Only limited information is available pertaining to the mechanism of DNA unwinding by RecQ helicases, and no crystallographic data are currently available. Biochemical studies of the prototype member, Escherichia coli RecQ helicase, indicate that the enzyme utilizes multiple interacting ATP-binding sites to unwind double-stranded DNA (7Harmon F.G. Kowalczykowski S.C. J. Biol. Chem. 2001; 276: 232-243Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The human WRN gene product, which is defective in the premature aging disorder Werner syndrome, encodes a 1,432 amino acid protein with the seven conserved motifs found in the RecQ family of Super-family 2 DNA helicases (8Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1479) Google Scholar). WRN 1The abbreviations used are: WRN, Werner Syndrome; PAH, polycyclic aromatic hydrocarbon; BcPh, benzo[c]phenanthrene; DE, 3,4-diol 1,2-epoxide; dA, deoxyadenosine.1The abbreviations used are: WRN, Werner Syndrome; PAH, polycyclic aromatic hydrocarbon; BcPh, benzo[c]phenanthrene; DE, 3,4-diol 1,2-epoxide; dA, deoxyadenosine. (9Shen J.C. Gray M.D. Oshima J. Loeb L.A. Nucleic Acids Res. 1998; 26: 2879-2885Crossref PubMed Scopus (181) Google Scholar), similar to all of the other RecQ helicases characterized to date, is a 3′–5′-helicase (reviewed in Refs. 4Mohaghegh P. Hickson I.D. Hum. Mol. Genet. 2001; 10: 741-746Crossref PubMed Scopus (188) Google Scholar and 10Shen J.C. Loeb L.A. Trends Genet. 2000; 16: 213-220Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Thus, WRN is thought to translocate 3′–5′ along the bound single-stranded DNA residing between the duplex portions of a helicase directionality substrate. However, unidirectional translocation of WRN on a DNA lattice remains to be demonstrated. Although the DNA substrate specificity of WRN (11Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and other RecQ helicases (12Bennett R.J. Keck J.L. Wang J.C. J. Mol. Biol. 1999; 289: 235-248Crossref PubMed Scopus (114) Google Scholar, 13Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (477) Google Scholar) has been studied, the effects of DNA damage on helicases of this family have not been characterized prior to this study.The replication (14Hanaoka F. Yamada M. Takeuchi F. Goto M. Miyamoto T. Hori T. Adv. Exp. Med. Biol. 1985; 190: 439-457Crossref PubMed Scopus (46) Google Scholar, 15Poot M. Hoehn H. Runger T.M. Martin G.M. Exp. Cell Res. 1992; 202: 267-273Crossref PubMed Scopus (186) Google Scholar, 16Salk D. Bryant E. Hoehn H. Johnston P. Martin G.M. Adv. Exp. Med. Biol. 1985; 190: 305-311Crossref PubMed Scopus (62) Google Scholar, 17Takeuchi F. Hanaoka F. Goto M. Akaoka I. Hori T. Yamada M. Miyamoto T. Hum. Genet. 1982; 60: 365-368Crossref PubMed Scopus (69) Google Scholar) and recombination (18Cheng R.Z. Murano S. Kurz B. Shmookler R.R. Mutat. Res. 1990; 237: 259-269Crossref PubMed Scopus (59) Google Scholar, 19Prince P.R. Emond M.J. Monnat Jr., R.J. Genes Dev. 2001; 15: 933-938Crossref PubMed Scopus (130) Google Scholar, 20Saintigny Y. Makienko K. Swanson C. Emond M.J. Monnat Jr., R.J. Mol. Cell. Biol. 2002; 22: 6971-6978Crossref PubMed Scopus (230) Google Scholar) defects observed in WRN–/– cells may reflect abnormal processing of specific structures associated with the replication fork or a DNA recombination intermediate. Our recent work demonstrated that WRN helicase is able to unwind a synthetic replication fork in the direction of the fork (11Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), a biochemical activity that may be important for the maintenance of fork progression. WRN was recently shown to interact functionally with DNA polymerase δ by stimulating the enzymatic rate of nucleotide incorporation (21Kamath-Loeb A.S. Johansson E. Burgers P.M. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4603-4608Crossref PubMed Scopus (156) Google Scholar). It was subsequently shown that WRN facilitates polymerase δ synthesis through hairpin and tetraplex structures that impede the polymerase, suggesting that WRN may remove secondary structure at a stalled replication fork to allow polymerase δ synthesis to proceed (22Kamath-Loeb A.S. Loeb L.A. Johansson E. Burgers P.M. Fry M. J. Biol. Chem. 2001; 276: 16439-16466Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The results to be reported here suggest that WRN may not function properly to ensure replication fork progression if the fork is blocked by a covalent DNA adduct, even though WRN may disrupt other DNA structures such as tetraplexes (13Mohaghegh P. Karow J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. Nucleic Acids Res. 2001; 29: 2843-2849Crossref PubMed Scopus (477) Google Scholar, 23Fry M. Loeb L.A. J. Biol. Chem. 1999; 274: 12797-12802Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) or triplexes (24Brosh Jr., R.M. Majumdar A. Desai S. Hickson I.D. Bohr V.A. Seidman M.M. J. Biol. Chem. 2000; 276: 3024-3030Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) that might impede a replication fork.Polycyclic aromatic hydrocarbon (PAH) 3,4-diol 1,2-epoxide (DE)-DNA adducts have been used by us as tools to probe the interactions between other DNA-processing enzymes such as topoisomerases (25Pommier Y. Laco G.S. Kohlhagen G. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10739-10744Crossref PubMed Scopus (50) Google Scholar, 26Tian L. Sayer J.M. Kroth H. Kalena G. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 9905-9911Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) and their DNA substrates as well as to elucidate possible molecular mechanisms for the induction of cancer by these DNA lesions. In this study, we examined the effects of site-specific benzo[c]phenanthrene (BcPh) DE adducts at N 6 of deoxyadenosine (Scheme I) positioned centrally in the double-stranded region of a forked DNA duplex substrate on the unwinding activity catalyzed by WRN helicase. These adducts are derived by both cis- and trans-opening of the (–)-(1R,2S,3S,4R)- and (+)-(1S,2R,3R,4S)-enantiomers of the DE in which the benzylic 4-hydroxyl group and epoxide oxygen are trans. The DEs are examples of mutagenic and carcinogenic bay-region diol epoxide metabolites that are formed from PAH by the combined action of cytochrome P-450 and epoxide hydrolase (27Jerina D.M. Sayer J.M. Agarwal S.K. Yagi H. Levin W. Wood A.W. Conney A.H. Pruess-Schartz D. Baird W.M. Pigott M.A. Dipple A. Kocsis J.J. Jollow D.J. Witmer C.M. Nelson J.O. Snyder R. Biological Reactive Intermediates III. Plenum Press, New York, NY1986: 11-30Google Scholar, 28Thakker D.R. Yagi H. Levin W. Wood A.W. Jerina D.M. Anders M.W. Bioactivation of Foreign Compounds. Academic Press, New York, NY1985: 177-242Google Scholar). Benzo[c]phenanthrene is a common environmental contaminant (29International Agency for Research on CancerPolynuclear Aromatic Compounds, Part I, Chemical, Environmental and Experimental Data, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. IARC, Lyon, France1983Google Scholar, 30International Agency for Research on Cancer. (1985) Polynuclear Aromatic Compounds, Part 4, Bitumens, Coal-Tars and Derived Products, Shale-Oils and Soots, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, IARC, Lyon, FranceGoogle Scholar, 31Bjorseth A. Jones P.W. Leber P. Third International Symposium on Chemistry and Biology: Carcinogenesis and Mutagenesis. Ann Arbor Science Publishers, Inc., Ann Arbor, MI1979: 371-381Google Scholar, 32de Vos R.H. van Dokkum W. Schouten A. de Jong-Berkhout P. Food Chem. Toxicol. 1990; 28: 263-268Crossref PubMed Scopus (205) Google Scholar, 33Lunde G. Bjorseth A. Nature. 1977; 268: 518-519Crossref PubMed Scopus (150) Google Scholar), which has been found in industrial emissions, wastewater, and food. Metabolism of the hydrocarbon on mouse skin (34Einolf H.J. Amin S. Yagi H. Jerina D.M. Baird W.M. Carcinogenesis. 1996; 17: 2237-2244Crossref PubMed Scopus (36) Google Scholar) by cultured rodent embryo cells (35Pruess-Schwartz D. Baird W.M. Yagi H. Jerina D.M. Pigott M.A. Dipple A. Cancer Res. 1987; 47: 4032-4037PubMed Google Scholar) or by human mammary carcinoma cells (34Einolf H.J. Amin S. Yagi H. Jerina D.M. Baird W.M. Carcinogenesis. 1996; 17: 2237-2244Crossref PubMed Scopus (36) Google Scholar) led to covalent BcPh DE-DNA adducts primarily at deoxyadenosine (dA). Notably, human liver microsomes metabolize BcPh via the carcinogenic diol epoxide pathway to a greater extent than do microsomes from rat liver (36Baum M. Amin S. Guengerich F.P. Hecht S.S. Kohl W. Eisenbrand G. Chem. Res. Toxicol. 2001; 14: 686-693Crossref PubMed Scopus (19) Google Scholar). The metabolically derived BcPh DEs react extensively with dA residues in DNA in vitro (37Dipple A. Pigott M.A. Agarwal S.K. Yagi H. Sayer J.M. Jerina D.M. Nature. 1987; 327: 535-536Crossref PubMed Scopus (171) Google Scholar) and in vivo (38Agarwal R. Canella K.A. Yagi H. Jerina D.M. Dipple A. Chem. Res. Toxicol. 1996; 9: 586-592Crossref PubMed Scopus (27) Google Scholar), and induce mutations at dA in mammalian cells (39Wei S.J. Chang R.L. Cui X.X. Merkler K.A. Wong C.Q. Yagi H. Jerina D.M. Conney A.H. Cancer Res. 1996; 56: 3695-3703PubMed Google Scholar). Such mutations provide an attractive mechanism for the induction of cell transformation leading to cancer. In particular the (–)-(1R,2S,3S,4R)-BcPh DE is one of the most tumorigenic PAH DEs identified to date (40Levin W. Chang R.L. Wood A.W. Thakker D.R. Yagi H. Jerina D.M. Conney A.H. Cancer Res. 1986; 46: 2257-2261PubMed Google Scholar).A particularly useful feature of the PAH DEs is the variety of structural motifs exhibited by the different DE adducts within duplex DNA. These structural motifs have been well characterized by NMR studies. The aromatic portions of the 1R and 1S trans-opened BcPh DE N 6-dA adducts used in this study (see Fig. 1) intercalate between adjacent base pairs in duplex DNA (41Cosman M. Fiala R. Hingerty B.E. Laryea A. Lee H. Harvey R.G. Amin S. Geacintov N.E. Broyde S. Patel D. Biochemistry. 1993; 32: 12488-12497Crossref PubMed Scopus (90) Google Scholar, 42Cosman M. Laryea A. Fiala R. Hingerty B.E. Amin S. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1995; 34: 1295-1307Crossref PubMed Scopus (93) Google Scholar). Local stretching of the double helix accommodates the intercalated aromatic rings, and the adducted adenine retains Watson-Crick base pairing to its complement within the helix despite some buckling and propeller twisting of the adducted base pair. The non-aromatic benzo-rings are situated in the major groove, and the intercalated aromatic portion projects toward but not into the minor groove. These trans-opened BcPh DE-dA adducts are oriented such that the aromatic moiety of the 1R dA adduct intercalates on the 5′ side of the adducted adenine base, whereas the aromatic moiety of the 1S dA adduct intercalates on the 3′ side (41Cosman M. Fiala R. Hingerty B.E. Laryea A. Lee H. Harvey R.G. Amin S. Geacintov N.E. Broyde S. Patel D. Biochemistry. 1993; 32: 12488-12497Crossref PubMed Scopus (90) Google Scholar, 42Cosman M. Laryea A. Fiala R. Hingerty B.E. Amin S. Geacintov N.E. Broyde S. Patel D.J. Biochemistry. 1995; 34: 1295-1307Crossref PubMed Scopus (93) Google Scholar). Although the solution structures of cis-opened BcPh DE-dA adducts in DNA have not been determined by NMR, the two cis-adducts in the present study are likely to exhibit the same dependence of the direction of intercalation on their configuration at C-1 as their trans-analogs.Fig. 1Inhibition of WRN helicase activity by a single BcPh DE adduct positioned in the strand on which WRN translocates is dependent on the stereochemistry of the adduct. Panel A, reaction mixtures (20 μl) containing 10 fmol of the indicated forked-duplex DNA substrate and specified concentrations of WRN were incubated at 37 °C for 15 min under standard conditions. Products were resolved on native 12% polyacrylamide gels. Phosphorimages of typical gels are shown. For each gel: lane 1, no enzyme (NE); lane 2, 0.6 nm WRN; lane 3, 1.2 nm WRN; lane 4, 2.4 nm WRN; lane 5, 4.8 nm WRN; lane 6, heat-denatured substrate control. Panel B, percent displacement from panel A (mean value ± S.D. of at least three experiments) indicated by error bars.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The present study demonstrates that cis- and trans-opened BcPh DE-dA adducts in DNA are highly effective probes of helicase activity. The cis-S BcPh DE-dA adduct in the strand on which WRN is presumed to translocate was a potent inhibitor of unwinding activity, but a forked duplex containing this adduct did not sequester WRN to any greater extent than an unmodified control. The trans-S adduct was not as effective as the cis-S isomer in inhibiting translocation. Inhibition of unwinding activity is also sensitive to the orientation of the adduct (toward or away from the advancing helicase) in the displaced strand of the duplex region, which is opposite to the strand on which the helicase translocates.MATERIALS AND METHODSProteins—Recombinant hexahistidine-tagged WRN protein (wild-type, WRN-E84A) was overexpressed using a baculovirus/Sf9 insect system and purified as described previously (43Orren D.K. Brosh Jr., R.M. Nehlin J.O. Machwe A. Gray M.D. Bohr V.A. Nucleic Acids Res. 1999; 27: 3557-3566Crossref PubMed Scopus (107) Google Scholar). UvrD (DNA helicase II) was overexpressed in Escherichia coli and purified as described previously (44Mechanic L.E. Frankel B.A. Matson S.W. J. Biol. Chem. 2000; 275: 38337-38346Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Purified recombinant hexahistidine-tagged BLM protein (45Karow J.K. Newman R.H. Freemont P.S. Hickson I.D. Curr. Biol. 1999; 9: 597-600Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) was kindly provided by Dr. Ian Hickson (University of Oxford). T4 polynucleotide kinase was obtained from New England Biolabs.Nucleotides, Oligonucleotides, and DNA Substrates—[3H]ATP was from Amersham Biosciences, and [γ-32P]ATP was from PerkinElmer Life Sciences. The oligonucleotides used for preparation of duplex DNA substrates are listed in Table I. Unadducted oligonucleotides were purchased from Lofstrand Technologies or Midland Certified Reagent Company. Oligonucleotide 25-mers containing diastereomerically pure cis- and trans-opened BcPh DE-dA adducts (Scheme I) were synthesized as their 5′-phosphates using a semi-automated procedure, essentially as described previously (Ref. 46Kroth H. Yagi H. Sayer J.M. Kumar S. Jerina D.M. Chem. Res. Toxicol. 2001; 14: 708-719Crossref PubMed Scopus (28) Google Scholar and references therein), with a manual step for coupling of the BcPh DE-dA-adducted phosphoramidites (47Ilankumaran P. Pannell L.K. Gebreselassie P. Pilcher A.S. Yagi H. Sayer J.M. Jerina D.M. Chem. Res. Toxicol. 2001; 14: 1330-1338Crossref PubMed Scopus (10) Google Scholar) as a mixture of their 1R/1S diastereomers. For details of the syntheses and chromatographic separation of the diastereomeric R/S pairs of cis- and trans-adducted oligonucleotides, see Supplemental Data. Absolute configurations were assigned to the separated (1R and 1S) oligonucleotide diastereomers by enzymatic hydrolysis (48Sayer C.M. Chadha A. Agarwal S.K. Yeh H.J. Yagi H. Jerina D.M. J. Org. Chem. 1991; 56: 20-29Crossref Scopus (130) Google Scholar) to the nucleoside adducts whose circular dichroism (CD) spectra were then compared with the known CD spectra (49Agarwal D. Sayer J.M. Yeh H.J.C. Pannell L.K. Hilton B.D. Piggot M.A. Dipple A. Yagi H. Jerina D.M. J. Am. Chem. Soc. 1987; 109: 2497-2504Crossref Scopus (149) Google Scholar) of the optically active BcPh DE-dA adducts. DNA duplex substrates were prepared as described previously (11Brosh Jr., R.M. Waheed J. Sommers J.A. J. Biol. Chem. 2002; 277: 23236-23245Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and are shown in Table II.Table IOligonucleotide sequences for DNA substratesTable IOligonucleotide sequences for DNA substratesTable IIDNA substratesTable IIDNA substratesHelicase Assays—Helicase assay reaction mixtures (20 μl) contained 30 mm Hepes (pH 7.6), 5% glycerol, 40 mm KCl, 0.1 mg/ml bovine serum albumin, 8 mm MgCl2, 2 mm ATP, 10 fmol of DNA duplex substrate (0.5 nm DNA substrate concentration), and the indicated concentrations of WRN, BLM, or UvrD. Helicase reactions were initiated by the addition of the respective helicase and then incubated at 37 °C for 15 min. Reactions were quenched with 10 μl of loading buffer (50 mm EDTA, 40% glycerol, 0.1% bromphenol blue, 0.1% xylene cyanol) containing a 10-fold excess of unlabeled oligonucleotide with the same sequence as the labeled strand. The products of the helicase reactions were resolved on nondenaturing 12% (19:1 acrylamide: bisacrylamide) polyacrylamide gels. Radiolabeled DNA species in polyacrylamide gels were visualized using a PhosphorImager and quantitated using the ImageQuant software (Molecular Dynamics). The percent helicase substrate unwound was calculated by the formula: percent unwinding = 100 × (P/(S + P)), where P is the product and S is the residual substrate. The values of P and S have been corrected after subtracting background values in controls containing no enzyme and heat-denatured substrate, respectively. Helicase data represent the mean of at least three independent experiments with mean ± S.D. shown by error bars.For helicase sequestration studies, WRN (3.6 nm, 72 fmol) was preincubated with the indicated amounts (0–500 fmol) of “unlabeled” single-stranded DNA (oligonucleotides unadducted A or adducted E) or “unlabeled” forked-duplex DNA molecules (substrates 1-unadducted or 5-cis-S) in standard helicase reaction buffer (described above) containing 2 mm ATP for 3 min at 24 °C. 10 fmol of radiolabeled forked-duplex molecule (11-tracker) was subsequently added to the reaction mixture and incubated for 7 min at 37 °C. Reactions were then quenched and resolved on native polyacrylamide gels as described above. Typically, 75–90% of the tracker-11 helicase substrate was unwound in reactions lacking the competitor DNA molecule. Displacement (% Control) is expressed relative to the control reactions lacking the competitor DNA.ATPase Assays—ATPase assay reaction mixtures (30 μl) contained 30 mm Hepes (pH 7.6), 5% glycerol, 40 mm KCl, 0.1 mg/ml bovine serum albumin, 8 mm MgCl2, the indicated duplex DNA effector concentration, 0.8 mm [3H]ATP, and 55 nm WRN. Reactions were initiated by the addition of WRN and incubated at 37 °C. Samples (5 μl) were removed at 2-min intervals and evaluated by thin layer chromatography as described previously (50Matson S.W. Richardson C.C. J. Biol. Chem. 1983; 258: 14009-14016Abstract Full Text PDF PubMed Google Scholar). <20% of the substrate ATP was consumed in the reaction over the entire time course (10 min) of the experiments. K eff values (Table III) were determined using linear regression analysis from double reciprocal plots of ATP hydrolysis initial rate versus forked-duplex DNA effector concentration (Fig. 4).Table IIIHydrolysis of ATP by WRN in presence of forked-duplex DNA effectorsTable IIIHydrolysis of ATP by WRN in presence of forked-duplex DNA effectorsFig. 4WRN requires similar concentrations of BcPh DE-modified and unmodified forked duplex for half-maximal ATP hydrolysis activity. DNA-stimulated ATP hydrolysis reactions were as described under “Materials and Methods” using 55 nm WRN, 0.8 mm [3H]ATP, and the indicated concentration of 1-unadducted (•)or5-cis-S (○) forked duplex as the DNA effector.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSTo understand better the effects of DNA structural elements on WRN-unwinding activity, we investigated how the helicase is affected by a series of structurally related single covalent DNA adducts. Specifically, the helicase substrates are forked DNA duplexes with a covalently bonded site-specific BcPh DE adduct in one of the two DNA strands (Table I) and positioned centrally in the 20-mer duplex region (Table II). For the purposes of this study, the stereochemistry and position of the adduct within the DNA duplex were varied. In the first class of substrates (Table II, substrates 1–5 with a 5-nucleotide 3′-unduplexed tail), the adduct is positioned within the shorter 25-mer strand on which WRN is presumed to 3′–5′ translocate. The second class of substrates (Table II, substrates 6–10 with a 25-nucleotide 3′-unduplexed tail) contains the same adducts as the first class, but the adduct is on the 25-mer strand opposite to the 45-mer strand on which WRN translocates. Because the forked-duplex substrates used in this study contain a relatively short duplex tract (20 bp), we were able to study WRN helicase activity by measuring the release of the labeled intact 45-mer in the absence of an auxiliary factor such as replication protein A. Our choice of forked-duplex DNA substrates harboring a site-specific BcPh DE adduct enabled us to assess the effects of adduct stereochemistry, orientation, and strand occupation on WRN helicase activity.Inhibition of WRN Helicase Activity by a Single BcPh DE-dA Adduct in the Strand on Which WRN Translocates—We first tested helicase substrates with the single BcPh DE-dA adduct positioned centrally in the duplex tract on the strand on which WRN translocates (substrates 1–5). For all of the five substrates tested, the percent duplex DNA substrate unwound depended on the concentration of WRN present in the reaction (Fig. 1). For both the adducted and unadducted forked-duplex (20 bp) substrates, WRN exonuclease activity at the blunt end of the DNA substrate was minimal (<2%) as evidenced by the appearance of an intact released oligonucleotide on native gels (Fig. 1A) and confirmed by analysis of products on urea-denaturing gels (data not shown). Similar DNA unwinding of substrate 1-unadducted by an exonuclease-defective mutant WRN protein (WRN-E84A) was also observed (data not shown). These results are consistent with a previous observation that displacement of short (16 and 22 bp) duplex tracts by WRN helicase activity is more rapid than digestion by the WRN exonuclease activity (51Opresko P.L. Laine J.P. Brosh Jr., R.M. Seidman M.M. Bohr V.A. J. Biol. Chem. 2001; 276: 44677-44687Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).Significant inhibition of WRN helicase activity by all four BcPh DE adducts was detected at all of the WRN concentrations tested, and the extent of helicase inhibition depended on the stereochemistry of the specific adduct (Fig. 1). Notably, the cis-opened adducts inhibited WRN unwinding more effectively than the trans-adducts (Fig. 1B). This difference was statistically significant at WRN protein concentrations of 2.4 and 4.8 nm. At a WRN concentration of 2.4 nm (Fig. 1B), the 5-cis-S adduct profoundly inhibited helicase" @default.
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• W2010606793 date "2003-10-01" @default.
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• W2010606793 title "Inhibition of Werner Syndrome Helicase Activity by Benzo[c]phenanthrene Diol Epoxide dA Adducts in DNA Is Both Strand-and Stereoisomer-dependent" @default.
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