Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

Artigo Acesso aberto Revisado por pares

Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro

2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês

10.1074/jbc.m310025200

ISSN

1083-351X

Autores

Vanessa C. Csitkovits, Ellen L. Zechner,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

The IncF plasmid protein TraI functions during bacterial conjugation as a site- and strand-specific DNA transesterase and a highly processive 5′ to 3′ DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5′ to 3′ unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5′ tail of single-stranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of single-stranded DNA of defined length was created and its structure verified. We found that substrates containing ≥27 nucleotides of single-stranded DNA 5′ to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands. The IncF plasmid protein TraI functions during bacterial conjugation as a site- and strand-specific DNA transesterase and a highly processive 5′ to 3′ DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5′ to 3′ unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5′ tail of single-stranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of single-stranded DNA of defined length was created and its structure verified. We found that substrates containing ≥27 nucleotides of single-stranded DNA 5′ to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands. Helicases are ubiquitous enzymes involved in nucleic acid metabolism (for review, see Refs. 1.Delagoutte E. von Hippel P.H. Q. Rev. Biophys. 2002; 35: 431-478Crossref PubMed Scopus (147) Google Scholar, 2.Delagoutte E. von Hippel P.H. Q. Rev. Biophys. 2003; 36: 1-69Crossref PubMed Scopus (128) Google Scholar, 3.Caruthers J.M. McKay D.B. Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (455) Google Scholar, 4.Marians K.J. Structure Fold. Des. 2000; 8: 227-235Abstract Full Text Full Text PDF Scopus (34) Google Scholar). The TraI protein of IncF plasmids (first characterized as DNA helicase I of Escherichia coli (5.Geider K. Hoffmann-Berling H. Annu. Rev. Biochem. 1981; 50: 233-260Crossref PubMed Scopus (166) Google Scholar)) is essential for the transmission of bacterial genes during conjugation (6.Willetts N. McIntire S. Contrib. Microbiol. Immunol. 1979; 6: 137-145PubMed Google Scholar, 7.Matson S.W. Sampson J.K. Byrd D.R. J. Biol. Chem. 2001; 276: 2372-2379Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In that process a copy of a conjugative plasmid is transferred unidirectionally in single-stranded form from one bacterial cell to another (for reviews, see Refs. 8.Zechner E.L. de la Cruz F. Eisenbrandt R. Grahn A.M. Koraimann G. Lanka E. Muth G. Pansegrau W. Thomas C.M. Wilkins B.M. Zatyka M. Thomas C.M. The Horizontal Gene Pool: Bacterial Plasmids and Gene Spread. Harwood Academic Publishers, Amsterdam2000: 87-174Google Scholar and 9.Firth N. Ippen-Ihler K. Skurray R.A. Neidhard F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbager H.E. Escherichia coli and Salmonella. Vol. 2. American Society for Microbiology, Washington, D. C.1996: 2377-2401Google Scholar). The TraI protein of IncF plasmids contributes to conjugative transfer in several ways. It is a component of a nucleoprotein complex, the relaxosome, which assembles with site specificity at the plasmid origin of transfer (oriT). Relaxosomes initiate the series of DNA-processing reactions that prepare plasmid DNA for transfer to a recipient cell. They are common to all self-transmissible and mobilizable plasmids in different degrees of complexity (for reviews, see Refs. 8.Zechner E.L. de la Cruz F. Eisenbrandt R. Grahn A.M. Koraimann G. Lanka E. Muth G. Pansegrau W. Thomas C.M. Wilkins B.M. Zatyka M. Thomas C.M. The Horizontal Gene Pool: Bacterial Plasmids and Gene Spread. Harwood Academic Publishers, Amsterdam2000: 87-174Google Scholar, 10.Pansegrau W. Lanka E. Prog. Nucleic Acid Res. Mol. Biol. 1996; 54: 197-251Crossref PubMed Google Scholar, and 11.Francia V.M. Varsaki A. Garcillán-Barcia M.P. Latorre A. Drainas C. de la Cruz F. FEMS Microbiol. Rev. 2003; (in press)Google Scholar). The simplest systems employ a plasmid-encoded DNA transesterase, or relaxase, protein that acts on a specific phosphodiester bond, nic, in the transfer origin. Cleavage at this site provides a point of origin for the directed 5′ to 3′ transmission of the plasmid genome to a recipient cell. Relaxase proteins are also active in relaxosomes containing auxiliary DNA-binding proteins of host or plasmid origin. IncF, IncW, IncP, and IncQ systems offer well studied examples (12.Kline B.C. Helinski D.R. 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Meyer R. J. Bacteriol. 1996; 178: 5762-5767Crossref PubMed Google Scholar, 21.Zhang S. Meyer R.J. Mol. Microbiol. 1997; 25: 509-516Crossref PubMed Scopus (56) Google Scholar). Among IncF plasmids factors known to stimulate nic cleavage include E. coli integration host factor and plasmid proteins TraM and TraY (22.Everett R. Willetts N. J. Mol. Biol. 1980; 136: 129-150Crossref PubMed Scopus (70) Google Scholar, 23.Inamoto S. Fukuda H. Abo T. Ohtsubo E. J. Biochem. (Tokyo). 1994; 116: 838-844Crossref PubMed Scopus (25) Google Scholar, 24.Nelson W.C. Howard M.T. Sherman J.A. Matson S.W. J. Biol. Chem. 1995; 270: 28374-28380Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 25.Howard M.T. Nelson W.C. Matson S.W. J. Biol. Chem. 1995; 270: 28381-28386Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26.Kupelwieser G. Schwab M. Högenauer G. Koraimann G. Zechner E.L. J. Mol. Biol. 1998; 275: 81-94Crossref PubMed Scopus (40) Google Scholar, 27.Karl W. Bamberger M. Zechner E.L. J. Bacteriol. 2001; 183: 909-914Crossref PubMed Scopus (24) Google Scholar). In the case of IncW plasmid R388, TrwA protein performs this role (13.Moncalián G. Grandoso G. Llosa M. de la Cruz F. J. Mol. Biol. 1997; 270: 188-200Crossref PubMed Scopus (54) Google Scholar, 14.Moncalián G. Valle M. Valpuesta J. de la Cruz F. Mol. Microbiol. 1999; 31: 1643-1652Crossref PubMed Scopus (23) Google Scholar). The IncF system and the functionally analogous IncW-IncN family of DNA-mobilizing systems are (thus far) unique in that they additionally specify a DNA helicase activity essential for conjugative DNA transmission. The DNA helicase activities are located in C-terminal domains of the bifunctional F TraI and R388 TrwC proteins (28.Abdel-Monem M. Taucher-Scholz G. Klinkert M.Q. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4659-4663Crossref PubMed Scopus (57) Google Scholar, 29.Grandoso G. Llosa M. Zabala J.C. de la Cruz F. Eur. J. Biochem. 1994; 226: 403-412Crossref PubMed Scopus (51) Google Scholar, 30.Matson S.W. Morton B.S. J. Biol. Chem. 1991; 266: 16232-16237Abstract Full Text PDF PubMed Google Scholar, 31.Reygers U. Wessel R. Müller H. Hoffmann Berling H. EMBO J. 1991; 10: 2689-2694Crossref PubMed Scopus (61) Google Scholar, 32.Sherman J.A. Matson S.W. J. Biol. Chem. 1994; 269: 26220-26226Abstract Full Text PDF PubMed Google Scholar, 33.Traxler B.A. Minkley Jr., E.G. J. Mol. Biol. 1988; 204: 205-209Crossref PubMed Scopus (59) Google Scholar). For reasons that are poorly understood, efficient gene transfer requires physical linkage of the relaxase and helicase domains (7.Matson S.W. Sampson J.K. Byrd D.R. J. Biol. Chem. 2001; 276: 2372-2379Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 34.Llosa M. Grandoso G. Hernando M.A. de la Cruz F. J. Mol. Biol. 1996; 264: 56-67Crossref PubMed Scopus (65) Google Scholar). This bifunctional arrangement of DNA transesterase and helicase activities is shared by adeno-associated virus replication initiation (Rep) 1The abbreviations used are: Repreplication initiation proteinsntnucleotide(s)dsDNAdouble-stranded DNAssDNAsingle-stranded DNAMOPS3-(N-morpholino)propanesulfonic acidDTTdithiothreitol. proteins (35.Im D.S. Muzyczka N. Cell. 1990; 61: 447-457Abstract Full Text PDF PubMed Scopus (360) Google Scholar). As revealed by very recent structural data, the endonuclease domain of adeno-associated virus-5 Rep proteins (36.Hickman A.B. Ronning D.R. Kotin R.M. Dyda F. Mol. Cell. 2002; 10: 327-337Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) bear the closest structural and functional relatedness to the N-terminal DNA transesterase domains of the F TraI and R388 TrwC proteins (37.Datta S. Larkin C. Schildbach J.F. Structure. 2003; 11 (in press)Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). 2A. Guasch, M. Lucas, G. Moncalian, M. Cabezas, R. Perez-Luque, F. X. Gomis-Ruth, F. de la Cruz Fd, and M. Coll, submitted for publication. Adeno-associated virus-2 Rep78 protein and an alternative splicing product, Rep68, bind the viral replicative form origin of replication, cleave the target DNA in a site and strand-specific manner, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. The N-terminal 224 amino acids of Rep78/68 (39.Owens R.A. Weitzman M.D. Kyostio S.R. Carter B.J. J. Virol. 1993; 67: 997-1005Crossref PubMed Google Scholar) are involved in interaction with recognition sequences (resolution binding sites) in the viral terminal repeats and in the viral integration site on human chromosome 19 (40.Linden R.M. Ward P. Giraud C. Winocour E. Berns K.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11288-11294Crossref PubMed Scopus (251) Google Scholar). This protein cleaves with site and strand specificity the terminal resolution site, trs, near resolution binding sites. The helicase activity of Rep78/68 has 3′ to 5′ polarity and is believed to assist the initiation of adeno-associated virus DNA replication at several stages including modifying the terminal repeat hairpin before nicking at trs, conversion of the terminal repeat region from the linear to hairpin form, and finally, possibly as a replicative helicase during synthesis of the viral genome (41.Zhou X. Zolotukhin I. Im D.S. Muzyczka N. J. Virol. 1999; 73: 1580-1590Crossref PubMed Google Scholar). replication initiation proteins nucleotide(s) double-stranded DNA single-stranded DNA 3-(N-morpholino)propanesulfonic acid dithiothreitol. The conjugative helicases initiate duplex unwinding unidirectionally from the plasmid transfer origin after strand-specific cleavage at nic by their N-terminal relaxase domains. The molecular mechanisms of these early steps of plasmid strand transfer remain poorly understood. Genetic and biochemical approaches have characterized the role of auxiliary factors in assisting the nic-specific cleavage reaction as well as the contribution of origin DNA sequence and topology (23.Inamoto S. Fukuda H. Abo T. Ohtsubo E. J. Biochem. (Tokyo). 1994; 116: 838-844Crossref PubMed Scopus (25) Google Scholar, 24.Nelson W.C. Howard M.T. Sherman J.A. Matson S.W. J. Biol. Chem. 1995; 270: 28374-28380Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 25.Howard M.T. Nelson W.C. Matson S.W. J. Biol. Chem. 1995; 270: 28381-28386Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26.Kupelwieser G. Schwab M. Högenauer G. Koraimann G. Zechner E.L. J. Mol. Biol. 1998; 275: 81-94Crossref PubMed Scopus (40) Google Scholar, 27.Karl W. Bamberger M. Zechner E.L. J. Bacteriol. 2001; 183: 909-914Crossref PubMed Scopus (24) Google Scholar, 42.Tsai M.M. Fu Y.H. Deonier R.C. J. Bacteriol. 1990; 172: 4603-4609Crossref PubMed Google Scholar, 43.Luo Y. Gao Q. Deonier R.C. Mol. Microbiol. 1994; 11: 459-469Crossref PubMed Scopus (41) Google Scholar, 44.Rice P.A. Yang S. Mizuuchi K. Nash H.A. Cell. 1996; 87: 1295-1306Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar). Nonetheless, the product of that reaction, the open circular form, is not directly accessible to the duplex unwinding activity of the physically linked DNA helicase domain. Thus, although recruitment of the conjugative helicase to oriT is achieved by site-specific recognition of the N-terminal relaxase domain and/or via interaction with additional oriT binding auxiliary proteins, the reaction as reconstituted thus far fails to effectively load the helicase at nic (45.Byrd D.R. Matson S.W. Mol. Microbiol. 1997; 25: 1011-1022Crossref PubMed Scopus (98) Google Scholar). In the cell, then, the relaxosome probably acquires a higher order structure in a staged initiation process that triggers helicase activation, similar perhaps to the orchestrated unwinding of origin DNA during replication initiation in prokaryotic and eukaryotic cells (46.Konieczny I. EMBO Rep. 2003; 4: 37-41Crossref PubMed Scopus (47) Google Scholar). Purified TraI and TrwC proteins do not exhibit sequence specificity for binding and translocating (5′ to 3′) on ssDNA (29.Grandoso G. Llosa M. Zabala J.C. de la Cruz F. Eur. J. Biochem. 1994; 226: 403-412Crossref PubMed Scopus (51) Google Scholar, 47.Kuhn B. Abdel-Monem M. Krell H. Hoffmann-Berling H. J. Biol. Chem. 1979; 254: 11343-11350Abstract Full Text PDF PubMed Google Scholar, 48.Lahue E.E. Matson S.W. J. Biol. Chem. 1988; 263: 3208-3215Abstract Full Text PDF PubMed Google Scholar). Reconstitution of the duplex-unwinding activity in vitro requires a region of ssDNA adjacent to the duplex (49.Abdel-Monem M. Lauppe H.F. Kartenbeck J. Durwald H. Hoffmann-Berling H. J. Mol. Biol. 1977; 110: 667-685Crossref PubMed Scopus (35) Google Scholar). In early work Hoffmann-Berling and co-workers (47.Kuhn B. Abdel-Monem M. Krell H. Hoffmann-Berling H. J. Biol. Chem. 1979; 254: 11343-11350Abstract Full Text PDF PubMed Google Scholar) estimated that the length of ssDNA required by TraI on the duplex 5′ end lies within an approximate range of 12–200 nt. At the time partial duplexes were generated by exonucleolytic erosion of flush termini, and technical limitations prevented a narrower definition of this requirement for TraI. To gain insights to the extent of localized duplex unwinding that may occur to start plasmid DNA strand transfer, we have investigated the length requirement for ssDNA within a linear duplex necessary for efficient TraI-catalyzed DNA unwinding. We find that single-stranded regions shorter than 27 nt do not support efficient helicase activity. Expression and Purification of R1 TraI—The expression construction pHP2 (50.Zechner E.L. Prüger H. Grohmann E. Espinosa M. Högenauer G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7435-7440Crossref PubMed Scopus (29) Google Scholar) carries the 5.3-kilobase pair traI gene of plasmid R1drd16 (GenBank™ accession number AY423546) under the control of the Ptac promoter in vector pGZ119HE (51.Lessl M. Balzer D. Lurz R. Waters V.L. Guiney D.G. Lanka E. J. Bacteriol. 1992; 174: 2493-2500Crossref PubMed Google Scholar). Cultures (600 ml) of E. coli SCS1[pHP2] were grown at 37 °C in 2× TY (16 g/liter Bacto-Tryptone, 10 g/liter Bacto yeast extract, 5 g/liter NaCl) medium (52.Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 433Google Scholar) supplemented with 0.1% (w/v) glucose, 25 mm MOPS (pH 8.0), 25 μg/ml thiamine/HCl, and 10 μg/ml chloramphenicol. At an A600 of 0.5, isopropyl-1-thio-α-d-galactopyranoside was added to a concentration of 1 mm. Shaking of cells was continued at 30 °C for 5 h. Cells were harvested by centrifugation and resuspended in a total volume of 100 ml of 20 mm spermidine, 200 mm NaCl, and 2 mm EDTA (pH 8.0) and frozen at –20 °C. Frozen cells were thawed overnight at 4 °C, and the suspension was adjusted to final concentrations of 50 mm Tris/HCl (pH 7.6), 30 mm NaCl, 5% (v/v) sucrose, 0.15% (v/v) Brij-58, 275 μg/ml lysozyme, and 0.1 mm phenylmethylsulfonyl fluoride. After 1 h at 0 °C the lysis mixture was centrifuged at 100,000 × g for 90 min at 4 °C. Solid ammonium sulfate was slowly added to the supernatant to 50% saturation (0.29 mg/ml), stirred for 30 min on ice, and then centrifuged 27,000 × g for 30 min at 4 °C. The precipitate was dissolved in a total volume of 40 ml in buffer A (20 mm Tris/HCl (pH 7.6), 10% glycerol (v/v), 0.1 mm EDTA, 1 mm DTT, and 50 mm NaCl). Fraction I was dialyzed against 3 changes of a 5-fold volume of buffer A for 3 h. The dialyzed fraction was loaded on a Pharmacia HiTrap™ heparin-Sepharose column. Proteins were eluted with a 0.05–1.2 m gradient of NaCl using buffer B (20 mm Tris/HCl (pH 7.6), 10% glycerol (v/v), 0.1 mm EDTA, 1 mm DTT, 1.2 m NaCl). TraI eluted at 415 mm NaCl (fraction II). Soluble ammonium sulfate was added to a final concentration of 1 m, and the mixture was applied to a phenyl-Sepharose column equilibrated with buffer C (20 mm Tris/HCl (pH 7.6), 10% glycerol (v/v), 0.1 mm EDTA, 1 mm DTT, and 1 m (NH4)2SO4). The protein was eluted using a decreasing gradient of 1 to 0 m (NH4)2SO4 in buffer D (20 mm Tris/HCl (pH 7.6), 10% glycerol (v/v), 0.1 mm EDTA, and 1 mm DTT). The TraI protein eluted at ∼250 mm (NH4)2SO4 (fraction III). Peak fractions were pooled, dialyzed against buffer D, concentrated against polyethylene glycol 20000 supplemented with glycerol to a final concentration of 40% (v/v), and stored at –20 °C (fraction IV). This fraction was greater than 90% pure TraI protein based on Coomassie-stained polyacrylamide-denaturing gels. Protein concentration was determined using the Bradford protein assay (Bio-Rad) with bovine serum albumin as standard. ATPase Assay—The γ-phosphohydrolase activity of partially purified protein fractions was determined in a 25-μl reaction mixture containing 25 fmol of circular single-stranded f1 DNA effector, 25 mm Tris/HCl (pH 7.5), 20 mm NaCl, 3 mm MgCl2, 5 mm β-mercaptoethanol, ∼0.66 pmol of [γ-32P]ATP (1.85 × 103 cpm/pmol), and 2 mm unlabeled ATP. Components were combined in a volume sufficient for 13 reactions and warmed to 30 °C. The reaction was started by the addition of ∼60 ng of protein/25-μl reaction mixture. Aliquots (25 μl) were removed at 0, 0.5, 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, and 20 min, and the reaction was stopped by the addition of ice-cold EDTA to 85 mm.6-μl aliquots were spotted on polyethyleneimine-cellulose TLC plates and developed in 1 m LiCl to separate inorganic phosphate from the mono-, di-, and triphosphates. The extent of ATP hydrolysis was quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics). Preparation of Helicase Substrates—Linear partial duplex substrates containing a single-stranded gap were prepared in several steps. Three dsDNA fragments (A, B, C) were generated in independent PCR reactions performed with 200 μm each dNTP, 20 ng of pBluescript II SK DNA (Stratagene), 0.2 μm each primer (Table I), and 1 unit of Dynazyme II polymerase (Finnzymes) in the buffer provided. To render one strand in each fragment sensitive to subsequent exonuclease attack, one primer in each pair was 5′-phosphorylated before amplification. The reverse primer used to amplify fragment A and the forward primers employed to create fragments B and C were phosphorylated nonradioactively by T4 polynucleotide kinase and reisolated using mini Quick Spin™ columns (Roche Applied Science). PCR products A and B were common to every substrate. Product C was unique for each because the site of annealing of the forward primer in each case was positioned a few nt further removed on the template pBluescript DNA relative to the reverse primer, which was constant for each preparation. Product C for all substrates was radiolabeled internally by adding [α-32P]dATP to the PCR reaction mixture. The conditions for amplification were 94 °C for 3 min then 30 cycles at 94 °C for 30 s, 60 °C for 40 s, and 72 °C for 1 min followed by one step at 72 °C for 5 min. For the 30ss and 40ss substrates 55 °C annealing temperature was required. The 5′-phosphorylated strand of each PCR fragment was then selectively degraded by 5 units of λ exonuclease III (New England Biolabs)/20-μl PCR reaction mix at 37 °C for 1 h. After nuclease treatment, the products were purified with Qiagen PCR purification kits. The yield and concentration of ssDNA were determined on 1.4% agarose Tris borate EDTA gels. To create the partial linear duplex DNA substrates, the three protected strands of fragments A, B, and C were combined at a molar ratio of 3:3:1 in 10 mm Tris/HCl (pH 8.5) and 200 mm NaCl and heat-denatured at 94 °C for 5 min. Annealing was achieved in reiterated cycles of decreasing temperature (1 °C over four 25-s increments per cycle) to a final temperature of 16 °C. This hybridization mix was used directly in helicase assays or stored at –20 °C.Table IOligonucleotides used in this studyA for5′-GCGGGCCTCTTCGCTATTACG-3′A rev5′-CCTGCGTTATCCCCTGATTCTGTG-3′B5′-CAATTCGCCCTATAGTGAGTCG-3′12ss5′-CCCCTCGAGGTCGACGG-3′16ss5′-TCGAGGTCGACGGTATCG-3′20ss5′-GGTCGACGGTATCGATAAGC-3′27ss5′-GGTATCGATAAGCTTGATATCG-3′30ss5′-ATCGATAAGCTTGATATCGAATTCC-3′40ss5′-TTGATATCGAATTCCTGCAGC-3′67ss5′-GATCCACTAGTTCTAGAGCG-3′ Open table in a new tab Verification of Partial Duplex Substrate Structure—Typical nuclease reaction mixtures (20 μl) contained 3–3.5 ng (375 pm) of intermediate products or hybridized helicase substrates and 2.5 units (S1 nuclease and HindII), 4 units (XhoI and NotI), 5 units (ApaI), or 7.5 units (PstI, PvuII, and HindIII) of enzyme. All enzymes and buffers were supplied by Takara. Incubation was at 37 °C for 1 h except S1 nuclease (30 min). The products were resolved with 1.4% agarose gels in Tris borate EDTA buffer at 7 V/cm for 2.5 h. Mixtures of dsDNA fragments of known length radioactively end-labeled using T4 polynucleotide kinase and [γ-32P]ATP were used as standards for gel mobility. Radiolabeled DNA species were visualized by autoradiography of the dried gels. Helicase Assay—Standard reactions (20 μl) were performed at 37 °C for 20 min in a solution containing 40 mm Tris/HCl (pH 7.5), 4 mm MgCl2, 1 mm DTT, 10% glycerol, 50 μg/ml bovine serum albumin, 1.8 mm ATP, ∼3–3.5 ng of DNA (375 pm) substrates, and 0–52 nm TraI. For kinetic studies carried out in the presence of 52 nm enzyme a scaled-up (14-fold) reaction mixture was assembled and warmed to 37 °C, and the reaction was started by the addition of protein. Portions (20 μl) were removed at 0.5, 1, 1.5, 2, 3, 4, 5, 7.5, 10, 12.5, 15, and 20 min, and the reactions were stopped by the addition of 0.2 volumes of loading buffer (50 mm EDTA, 50% glycerol, 1% SDS, and 0.1% bromphenol blue). The products were resolved on 1.4% agarose gels in Tris borate EDTA buffer at 7 V/cm for 2.5 h. Radiolabeled DNA was visualized by autoradiography of the dried gels. Data were quantified using ImageQuant software (Molecular Dynamics). The percent unwound helicase substrate was determined after background correction: % unwound = signal intensity in displaced fragment divided by total substrate signal. Expression, Purification, and ATPase Activity of R1 TraI Protein—TraI was overexpressed in E. coli SCS1 and purified from crude cell extracts as described under “Experimental Procedures.” The final fraction contained a greater than 90% homogeneous solution of TraI as judged by SDS-PAGE and Coomassie Blue staining (data not shown). The purified protein exhibited an apparent molecular mass of 180 kDa. Typically, 20–25 mg of protein were obtained from 4.8 liters of cultured cells. Extensive studies have characterized the NTPase activity of TraI from plasmid F (97% identity between predicted proteins) (53.Abdel-Monem M. Hoffmann-Berling H. Eur. J. Biochem. 1976; 65: 431-440Crossref PubMed Scopus (108) Google Scholar, 54.Abdel-Monem M. Dürwald H. Hoffmann-Berling H. Eur. J. Biochem. 1976; 65: 441-449Crossref PubMed Scopus (142) Google Scholar). In good agreement with these reports the ATPase activity of TraI from plasmid R1 was dependent on the presence of a ssDNA effector and activated by Mg2+. In the presence of 2 mm ATP maximal activity was observed at 2 mm MgCl2, and half-maximal values were obtained at 0.3 and 20 mm MgCl2 (not shown). One unit of enzymatic activity is defined as the amount of enzyme needed to hydrolyze 1 nm ATP in 20 min at 30 °C. The progress of the purification procedure was monitored by measuring the ATPase activity at the later stages, as summarized in Table II. A specific activity of 1970 kilounits/mg was determined for the final fraction containing TraI.Table IIPurification of R1TraIStepsVolumeTotal proteinSpecific activitymlmgkilounits/mgI. Cell extract1322117NDII. AS50 precipitation28535NDIII. Heparin-Sepharose48721010IV. Phenyl-Sepharose28231970 Open table in a new tab Preparation and Verification of the Helicase Substrate Structures—The TraI protein of plasmid F is known to require a region of ssDNA adjacent to the duplex to facilitate its DNA unwinding activity (47.Kuhn B. Abdel-Monem M. Krell H. Hoffmann-Berling H. J. Biol. Chem. 1979; 254: 11343-11350Abstract Full Text PDF PubMed Google Scholar, 48.Lahue E.E. Matson S.W. J. Biol. Chem. 1988; 263: 3208-3215Abstract Full Text PDF PubMed Google Scholar, 49.Abdel-Monem M. Lauppe H.F. Kartenbeck J. Durwald H. Hoffmann-Berling H. J. Mol. Biol. 1977; 110: 667-685Crossref PubMed Scopus (35) Google Scholar). To evaluate the requirement of this enzyme for a 5′ ssDNA tail, we chose to create a series of linear partial duplex substrates containing a centrally located single-stranded gap of variable length. Duplex arms were generated by hybridizing two distinct non-overlapping single-stranded fragments to one common longer fragment of complementary ssDNA as illustrated in Fig. 1. To be able to control the gap size precisely, the position of the 5′ end of the primer utilized to amplify fragment C was varied incrementally relative to the start of the left arm duplex (Fig. 1B). A series of partial duplex molecules were generated with this approach that differed only in the defined length of the single-stranded region present after hybridization. The structure of the resulting products was verified based on nuclease sensitivity (Fig. 2). The oriT region of IncF(II) plasmid R1 is essentially devoid of commonly used restriction endonuclease cleavage sites. Utilization of this DNA for creating suitable helicase substrates would make verification of the gap size in each substrate prohibitively difficult. Therefore, pBluescript DNA was chosen to be able to exploit the dense arrangement of restriction endonuclease recognition sites in the multiple cloning site when these are positioned in and around the single-stranded gap of the partial duplex hybridization products. For each substrate preparation sensitivity of intermediate products and the product of their hybridization to restriction endonucleases and single strand-specific nuclease S1 was analyzed. The results for three of the seven different substrates used in this study are presented in detail. In Fig. 2A the structure of the hybridization product expected to contain a single-stranded gap 67 nt in length is demonstrated. Importantly, for every substrate just one of the component ssDNA fragments was radiolabeled. Thus, not all DNA species are visible in the autoradiogram. The different DNA species present after λ-exonuclease III treatment of the pooled PCR products before their hybridization (lane 2) were also treated with several nucleases and resolved electrophoretically through non-denaturing agarose gels (lanes 3–6). The fastest and the slowest migrating labeled species were insensitive to restriction endonucleases (lanes 3–5) but were completely degraded by S1 nuclease (lane 6). In contrast, the band migrating somewhat less than the 405-bp double-stranded standard DNA fragment (lane 13) was resistant to S1 nuclease (lane 6). Treatment with PstI did not alter the appar

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Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro