The Phage T4 Protein UvsW Drives Holliday Junction Branch Migration
2007; Elsevier BV; Volume: 282; Issue: 47 Linguagem: Inglês
10.1074/jbc.m705913200
ISSN1083-351X
AutoresMichael R. Webb, Jody L. Plank, David T. Long, Tao‐shih Hsieh, Kenneth N. Kreuzer,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoThe phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair. The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair. Homologous recombination plays essential, but seemingly paradoxical, roles in promoting genetic diversity through meiotic recombination and in maintaining genomic stability. The importance of recombination in genomic stability is ensured by its roles in the repair of DNA damage such as double strand breaks and in the restart of stalled replication forks (for review, see Ref. (1McGlynn P. Lloyd R.G. Nat. Rev. Mol. Cell. Biol. 2002; 3: 859-870Crossref PubMed Scopus (362) Google Scholar)). For all of these processes, the Holliday junction (HJ) 2The abbreviations used are: HJ, Holliday junction; BM, branch migration; RDR, recombination-dependent replication; GST, glutathione S-transferase; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; ATPγS, adenosine 5′-O-(thiotriphosphate). is a common feature. HJs can be formed through an enzyme-mediated process that involves the invasion of a single-stranded portion of a DNA into a homologous sequence in another DNA. HJs may also be formed through regression of an inactive replication fork. The branch point of a HJ can migrate through strand exchange, as long as both participating duplex segments are homologous. Branch migration (BM) has been shown to be catalyzed by a number of enzymes including RecG (2Whitby M.C. Vincent S.D. Lloyd R.G. EMBO J. 1994; 13: 5220-5228Crossref PubMed Scopus (99) Google Scholar) and RuvAB (3Tsaneva I.R. Muller B. West S.C. Cell. 1992; 69: 1171-1180Abstract Full Text PDF PubMed Scopus (212) Google Scholar) from Escherichia coli, a subset of eukaryotic RecQ enzymes (4Plank J.L. Wu J. Hsieh T.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11118-11123Crossref PubMed Scopus (124) Google Scholar, 5Karow J.K. Constantinou A. Li J.L. West S.C. Hickson I.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6504-6508Crossref PubMed Scopus (423) Google Scholar), and Rad54 from human cells (6Bugreev D.V. Mazina O.M. Mazin A.V. Nature. 2006; 442: 590-593Crossref PubMed Scopus (144) Google Scholar). Bacteriophage T4 has been used as a model biological system since the early days of molecular biology and has been invaluable in advancing our understanding of many fundamental biological processes. The T4 genome encodes ∼300 different proteins, including all essential enzymes at the replication fork, making it a relatively simple organism to study (7Miller E.S. Kutter E. Mosig G. Arisaka F. Kunisawa T. Ruger W. Microbiol. Mol. Biol. Rev. 2003; 67 (Table of contents): 86-156Crossref PubMed Scopus (555) Google Scholar). Furthermore, T4 proteins show about as much sequence homology to eukaryotes as they do to prokaryotes (8Bernstein H. Bernstein C. J. Bacteriol. 1989; 171: 2265-2270Crossref PubMed Google Scholar). For example, T4 DNA polymerase (gp43) shows strong homology to the B family DNA polymerases of the archaeal, eucaryal, and viral kingdoms (9Spicer E.K. Rush J. Fung C. Reha-Krantz L.J. Karam J.D. Konigsberg W.H. J. Biol. Chem. 1988; 263: 7478-7486Abstract Full Text PDF PubMed Google Scholar); T4 thymidylate synthase and the replisome sliding clamp protein (gp45) show extensive conserved regions with the corresponding proteins from Bacteria and Eucary; and a subunit of the DNA polymerase clamp loader, gp44, is homologous to eukaryotic replication factor C (7Miller E.S. Kutter E. Mosig G. Arisaka F. Kunisawa T. Ruger W. Microbiol. Mol. Biol. Rev. 2003; 67 (Table of contents): 86-156Crossref PubMed Scopus (555) Google Scholar). Replication proceeds through two distinct pathways in T4 infections (10Kreuzer K.N. Trends Biochem. Sci. 2000; 25: 165-173Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Origin-dependent replication initiates at specific replication origins in the early stages of infection by assembly of replication complexes onto persistent RNA-DNA hybrids (R-loops). Recombination-dependent replication (RDR) predominates later in infection, and involves the assembly of replication fork complexes onto D-loop recombination intermediates. The same enzymes involved in RDR are also involved in double-stranded DNA break repair, and thus the processes of recombination, replication and DNA repair are all tightly interconnected (11George J.W. Stohr B.A. Tomso D.J. Kreuzer K.N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8290-8297Crossref PubMed Scopus (32) Google Scholar, 12Kreuzer K.N. Annu. Rev. Microbiol. 2005; 59: 43-67Crossref PubMed Scopus (77) Google Scholar). The T4 UvsW gene was first identified through mutant phenotypes which included increased sensitivity to hydroxyurea, decreased recombination, increased sensitivity to UV, and decreased UV mutability (13Derr L.K. Kreuzer K.N. J. Mol. Biol. 1990; 214: 643-656Crossref PubMed Scopus (23) Google Scholar, 14Derr L.K. Drake J.W. Mol. Gen. Genet. 1990; 222: 257-264Crossref PubMed Scopus (15) Google Scholar). Some UvsW mutants had dissimilar effects on replication and double-strand break repair, indicating that UvsW might have distinct multiple functions that could be uncoupled (14Derr L.K. Drake J.W. Mol. Gen. Genet. 1990; 222: 257-264Crossref PubMed Scopus (15) Google Scholar, 15Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (68) Google Scholar). Subsequently, it was shown that the UvsW protein could unwind R-loops in an ATP-dependent manner in vitro. Furthermore, expression of UvsW at late times of infection represses origin-dependent replication presumably by unwinding the origin R-loop intermediate (16Dudas K.C. Kreuzer K.N. Mol. Cell. Biol. 2001; 21: 2706-2715Crossref PubMed Scopus (41) Google Scholar). In addition, UvsW protein was found to possess a branched-DNA specific helicase activity that was ATP-dependent and abolished by the K141R mutation in the Walker A motif of a RecA-like domain of UvsW (15Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (68) Google Scholar). Subsequent x-ray crystallography of the N-terminal region of UvsW-K141R confirmed that the protein fits the structural profile of a superfamily II DNA helicase and suggested unique structural domains for DNA binding (17Sickmier E.A. Kreuzer K.N. White S.W. Structure. 2004; 12: 583-592Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). UvsW has also been shown to function as a 3′→5′ helicase and to promote single-stranded DNA annealing (18Nelson S.W. Benkovic S.J. J. Biol. Chem. 2007; 282: 407-416Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The UvsW protein can complement E. coli recG rnhA double mutants, most likely through unwinding of persistent R-loops in the bacterial chromosome (15Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (68) Google Scholar). Accordingly, the E. coli RecG helicase shows very similar functional properties to UvsW, including R-loop unwinding, branched-DNA specific unwinding, and a weak 3′ → 5′ helicase activity, though there is only limited structural homology between the two (17Sickmier E.A. Kreuzer K.N. White S.W. Structure. 2004; 12: 583-592Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 19McGlynn P. Lloyd R.G. Nucleic Acids Res. 1999; 27: 3049-3056Crossref PubMed Scopus (70) Google Scholar). RecG has been shown to promote BM of HJs in vitro, and to facilitate the regression of stalled replication forks (20McGlynn P. Lloyd R.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8227-8234Crossref PubMed Scopus (159) Google Scholar, 21Whitby M.C. Lloyd R.G. J. Biol. Chem. 1998; 273: 19729-19739Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Double-strand break repair is only partially reduced in T4 mutants lacking gp49 (Endo VII; HJ cleaving endonuclease), but is completely lost in the uvsW/49 double mutant (15Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (68) Google Scholar). This suggests that HJs can be processed by two different pathways in a T4 infection: Endo VII-catalyzed cleavage and a UvsW-promoted HJ resolving activity, most likely involving BM. To date, however, the enzyme(s) responsible for in vivo BM of HJs in T4 has not been identified, although the strand exchange enzyme UvsX and the helicase gp41 (in conjunction with its loader protein gp59) have been shown to promote 3-strand BM in vitro (22Salinas F. Kodadek T. Cell. 1995; 82: 111-119Abstract Full Text PDF PubMed Scopus (53) Google Scholar). The roles of UvsW protein in recombination, repair and mutagenesis, together with the similarities to RecG, suggest that UvsW could be a HJ BM enzyme. Here we confirm this prediction by showing that UvsW promotes the migration of a HJ through ∼1000 bp of DNA, using a novel HJ-containing substrate that should be generally useful. We also show that UvsW can resolve HJs and replication fork intermediates composed of T4-modified DNA generated during T4 infection. Enzymes—Dda helicase was a generous gift of Dr. Stephen W. White (St. Jude Children's Research Hospital, Memphis, TN). Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). RNase (DNase-free) was obtained from Roche Applied Sciences (Indianapolis, IN) and PreScission protease was obtained from GE Healthcare (Buckinghamshire, UK). Reverse gyrase was purified from Archaeoglobus fulgidus according to a published procedure (23Rodriguez A.C. J. Biol. Chem. 2002; 277: 29865-29873Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Expression Plasmid Construction—The plasmid pKCK47 carries the UvsW gene (GenBank™ accession AF158101.6; Tulane T4-like genome data base) as a GST fusion in a pGEX-3X expression vector (Amersham Biosciences) (15Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (68) Google Scholar). Repeated DNA sequencing of this construct as well as a PCR product of T4 genomic DNA consistently showed an AG → GA sequence inversion at T4 map position 114045 (update 3/28/2003). Thus, the data base sequence was incorrect, and the codon 457 should read GAC (aspartate) rather than AGC (serine). The plasmid pKCK48 carries a point mutation that causes amino acid substitution K141R in UvsW. To produce the inframe expression constructs described below, both plasmids were modified by insertion of a cytosine two bases upstream of the UvsW start codon using a QuikChange™ site-directed mutagenesis kit (Stratagene). The plasmids were then digested with BamHI, and the resulting UvsW- and UvsW-K141R-containing fragments were ligated to BamHI-digested plasmid pGEX-6p-1 (Amersham Biosciences) producing the UvsW-containing plasmid pMRW47-6p, and the K141R-containing plasmid, pMRW48-6p. These constructs generated glutathione S-transferase (GST)-UvsW fusions under control of the IPTG-inducible Ptac promoter. The linker region of pGEX-6p-1 encodes a unique amino acid sequence that is cleaved by Prescission protease, allowing the selective cleavage of GST from the fusion products. The residual peptide GPLGST remains attached to the N-terminal end of the purified proteins following cleavage. A version of pGEX-6p-1 that had its ColE1-based origin of replication replaced with an R6K γ replication origin was constructed as follows. Plasmid pGEX-6p-1 was first digested with AlwNI and PflMI, producing two fragments, the smaller of which contained only the ColE1-based replication origin and flanking, non-coding, sequences. To obtain an R6K γ replication origin sequence with PflMI- and AlwNI-ligatable ends, the R6K γ-containing plasmid pGPS4 (New England Biolabs) was used as a template for PCR amplification using primers flanking the origin sequence (position 369 through 678) but designed to produce PflMI- and AlwNI-digestible ends. The purified PCR product was then digested with PflMI and AlwNI, repurified, and ligated to the purified, large AlwNI/PflMI pGEX-6p-1 digestion fragment (minus the ColE1 replication origin). The resulting plasmid (pMRW3) was transformed into a π protein-expressing strain of E. coli, BW23322 (Δ(argF-lac)169, ΔuidA4::pir-116, rpoS396(Am), endA9(del-ins)::FRT, rph-1, hsdR514, creC510, robA1), selected with ampicillin and confirmed by DNA sequence analysis. To produce a UvsW-expressing version of pMRW3, both pMRW3 and pMRW47-6p were digested with BsaI and EcoNI. The gel-purified fragment containing the R6K γ replication origin from pMRW3 was then ligated to the UvsW-containing fragment of pMRW47-6p using T4 DNA ligase. The resulting plasmid (pMRW7) was obtained after transformation into BW23322 and confirmed by sequence analysis. UvsW Protein Purification—E. coli strain BW23322 containing the pMRW7 plasmid was grown at 37 °C in 1 liter of Luria broth (LB) containing carbenicillin (100 μg/ml) with vigorous shaking to an A600 of ∼1.0, at which point IPTG was added to 1 mm. After 1 h, cells were harvested by centrifugation at 4,000 × g (4 °C) and resuspended in 8 volumes of PBSX (10 mm Na2HPO4, 2 mm KH2HPO4, 3 mm KCl, 500 mm NaCl, pH 7.3) containing one Complete™ (Mini) Protease Inhibitor Mixture tablet (Roche Applied Science). Resuspended cells were frozen in dry ice/ethanol and either stored at -80 °C or used immediately as follows. After thawing, the cells were treated with Triton X-100 (0.2%) and lysozyme (1 mg/ml), refrozen and thawed, and then sonicated. Cell debris was removed by centrifugation at 45,000 × g for 20 min, followed by filtration through a 0.22-μm filter. ATP (2 mm) and Mg2+ (4 mm) were added to the homogenate which was incubated at 37 °C for 5 min. Glutathione-Sepharose 4B resin was then added and gently mixed for 1.5 h at 4 °C. This suspension was placed in a column, washed three times with PBSX, and then treated with RNase (5 μg) by mixing one bed volume of an RNase-TE buffer (10 mm Tris-HCl, pH 7.6; 0.25 mm EDTA) with the resin and incubating for 5 min at 37 °C. The column was then washed three times with HPB (290 mm Na2HPO4, 110 mm NaH2PO4, pH 7.2) and once with PPCB (50 mm Tris-HCl (pH 7.0), 150 mm NaCl, 1 mm EDTA, 1 mm DTT), and then PreScission protease (20 units in one bed volume of PPCB) was added, and the protease-containing column was incubated for 12 h at 4 °C. Cleaved protein was eluted with PBSX, and fractions were checked for purity using UV absorbance and SDS-PAGE with Coomassie staining. Selected fractions were dialyzed against 20 mm Tris (pH 7.6), 100 mm NaCl, 1 mm DTT, 0.5 mm EDTA, and 50% glycerol. Protein concentration was determined (Bio-Rad protein assay), and samples were stored at -20 °C. The overall yield was ∼600 μg. The UvsW-K141R protein was prepared as follows. A 0.5-liter LB culture of BL21/pLysS containing pMRW48-6p was grown to an A600 of ∼1.1 and induced with IPTG at 0.5 mm. The protein purification was essentially the same as that used for UvsW, except that K141R was eluted from the column using HPB. The overall yield was 1.4 mg. Oligonucleotide Substrates—DNA unwinding substrates (Table 1) were made by annealing the following oligonucleotides in the appropriate combinations, generally as described (24Brosh Jr., R.M. Opresko P.L. Bohr V.A. Methods Enzymol. 2006; 409: 52-85Crossref PubMed Scopus (32) Google Scholar, 25Constantinou A. West S.C. Methods Mol. Biol. 2004; 262: 239-253PubMed Google Scholar). A0 (5′-ACGCTGCCGAATTCTGGCTTGCTAAAGGATAGGTCGAATTTCTCATTTT-3′), B0 (5′-CAAAGTAAGAGCTTCTCGAGCTGCGCTAGCAAGCCAGAATTCGGCAGCGT-3′), C0 (5′-TCTTTGCCCAAATGCAGGTTCACCCGCGCAGCTCGAGAAGCTCTTACTTTG-3′), D0 (5′-AAAATGAGAAAATTCGACCTATCCTTGGGTGAACCTGCATTTGGGCAAAGA-3′), E0 (5′-AAAATGAGAAAATTCGACCTATCCTTGCGCAGCTCGAGAAGCTCTTACTTTG-3′), a2 (5′-AAGGATAGGTCGAATTTTCTCATTTT-3′), b2 (5′-TAGCAAGCCAGAATTCGGCAGCGT-3′), c2 (5′-GCGCAGCTCGAGAAGCTCTTACTTTG-3′), d1 (5′-AAATGAGAAAATTCGACCTATCCTT-3′). All oligonucleotides were purified by denaturing PAGE with gel extraction prior to annealing and, after annealing, by non-denaturing PAGE with electroelution and dialysis into 10 mm Tris-HCl (pH 7.6) containing 5 mm MgCl2. Appropriate oligonucleotides were 5′-end labeled with [γ-32P]ATP and polynucleotide kinase; only one strand of each substrate was labeled (indicated by *). The molar concentrations of the final DNA substrates were estimated by relating the specific activity of the labeled oligonucleotide to the activity of the purified substrates. Gel reference markers were generated by denaturing the appropriate substrate at 100 °C for 5 min. Oligonucleotide-based Unwinding Assay—Reactions were carried out in 20 μl of helicase reaction buffer (30 mm Tris-HCl (pH 7.5), 40 mm sodium acetate, 1 mm DTT, 5% glycerol, and 0.1 mg/ml bovine serum albumin) containing MgCl2 and ATP as indicated. Typically, substrates were added to yield concentrations of 0.5 nm (10 fmol per reaction), followed immediately by the addition of enzyme or control solution. Reactions were incubated at 37 °C for 15 min and then terminated by addition ofa2-μl stop buffer (100 mm Tris-HCl (pH 7.5), 50 mm EDTA, 2% SDS, 5 mg/ml proteinase K, 50% glycerol, 0.1% bromphenol blue, and 0.1% xylene cyanol) containing 50 nm of the unlabeled version of the substrate used for that particular reaction. The samples were incubated for 30 min at 30 °C and then separated on 7.5% polyacrylamide gels using 0.5× TBE (44.5 mm Tris-HCl, 44.5 mm borate, 1 mm disodium EDTA) at 4 °C (typically at 12.5 V/cm). Gels were fixed in 10% acetic acid/methanol, dried briefly, exposed to a PhosphorImager screen overnight and visualized using PhosphorImager and ImageQuant software (Molecular Dynamics). DNA-trioxsalen Cross-linking—DNA substrates (25-50 ng/μl) were treated with trioxsalen (final concentration 0.2 mm) and were exposed to a long-wave UV source for 20 min at room temperature. Double Holliday Junction Synthesis—The Double Holliday Junction Substrate 2 (DHJS-2) was synthesized utilizing methods developed by Plank and Hsieh (26Plank J.L. Hsieh T.S. J. Biol. Chem. 2006; 281: 17510-17516Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Briefly, A/B and B/A large heterodimers were prepared by annealing and linking purified ssDNA, using Archaeoglobus fulgidus reverse gyrase as described (27Plank J.L. Chu S.H. Pohlhaus J.R. Wilson-Sali T. Hsieh T.S. J. Biol. Chem. 2005; 280: 3564-3573Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The reactions were then stopped by the addition of EDTA to 10 mm and SDS to 1%, and incubated at 80 °C for an additional 5 min. KCl was added to 500 mm, and the solution was cooled on ice for 15 min to precipitate the KDS and protein. The precipitate was removed by centrifugation at 20,000 × g for 20 min, and the cleared supernatant containing the DNA was loaded onto a Qiagen DEAE column. The column was washed with a solution containing 10 mm MOPS (pH 7.0), 1 m NaCl, 4 m urea, and 30% ethanol to remove any remaining ssDNA circles (28Bregeon D. Doetsch P.W. BioTechniques. 2004; 37 (764, 766): 760-762Crossref PubMed Google Scholar), and the large heterodimers were then eluted per kit instructions (Qiagen, Inc.). The two large heterodimers were then annealed and linked to each other using the same reaction conditions as above. This reaction was then stopped with the addition of EDTA to 10 mm and SDS to 1%, and the DNA was extracted with phenol/chloroform and precipitated with ethanol. The substrate was then dissolved in 10 mm Tris (pH 7.9), 0.1 mm EDTA and spectrophotometrically quantified. Holliday Junction Branch Migration Assay—DHJS-2 was digested simultaneously with BamHI and AlwNI for 1 h at 30 °C in NEBuffer 2 (10 mm Tris-HCl (pH 7.9), 10 mm MgCl2, 50 mm NaCl, 1 mm DTT). BM assays were carried out at 37 °C using the digested DHJS-2 in 20 μl of helicase reaction buffer (30 mm Tris-HCl, pH 7.5, 40 mm sodium acetate, 1 mm DTT, 5% glycerol, and 0.1 mg/ml bovine serum albumin) containing MgCl2 and ATP (or analogue) at the concentration indicated. Typically, DNA substrate concentrations were 7.5-15 ng/μl and substrate addition was followed immediately by the addition of enzyme or control solution. Reactions were terminated by addition of 2 μl of (10×) stop buffer (20% Ficoll, 1% SDS, 100 mm MgCl2, 10 μg/ml ethidium bromide, 0.1% bromphenol blue, and 0.1% xylene cyanol) and 2 μl of proteinase K (5 mg/ml). Following incubation at 30 °C for 30 min, the products were separated on a 1.2% agarose gel containing 0.5 μg/ml ethidium bromide. Gels were run overnight at 4 °C at 4 V/cm, destained, and the DNA was visualized using an Alphaimager (Alpha Innotech Corp.). T4 Infection and DNA Purification—Bacterial cells (E. coli CAG12135 derivative: acrA::Tn10-kan, recA::Tn10-cam, and recD) were grown with vigorous shaking at 37 °C to an A560 of 0.5 (∼4 × 108 per ml), and infected with T4 strain K10-49am [amB262 (gene 38), amS29 (gene 51), nd28 (denA), rIIPT8 (rII-denB deletion), amE727 (gene 49)] at a multiplicity of 6 PFU per cell. After 4 min at 37 °C without shaking, cells were incubated for an additional 28 min with shaking. Infected cells from 1 ml of culture were collected by centrifugation at 13,000 rpm for 2 min, and immediately resuspended in 300 μl of SDS lysis buffer (50 mm Tris-HCl (pH 7.8), 10 mm EDTA, 100 mm NaCl, 0.2% SDS). Proteinase K was added to 0.5 mg/ml, and the suspension was incubated at 65 °C for 2 h. Total nucleic acid was extracted sequentially with phenol/chloroform-isoamyl alcohol (24:1), and dialyzed overnight at 4 °C against TE buffer (10 mm Tris-HCl, pH 7.8; 1 mm EDTA). Two-dimensional Gel Analysis of T4 Replication Intermediates—Purified DNA from T4 infections was treated with the restriction enzyme PacI in 1× restriction buffer (50 mm NaCl, 10 mm Tris-HCl (pH 7.8), 3.5 mm MgCl2, 1 mm DTT, 0.1 mg/ml bovine serum albumin) at 37 °C overnight. The restriction digestion reaction was then supplemented to give the following concentrations: 1 mm ATP, 20 mm Tris-HCl, pH 7.8, and 68 nm purified UvsW (unless otherwise indicated). Reactions were incubated at 37 °C for the indicated time, followed by sequential DNA extraction with phenol/chloroform-isoamyl alcohol (24:1). Two-dimensional agarose gel electrophoresis was carried out according to the procedure of Friedman and Brewer (29Friedman K.L. Brewer B.J. Methods Enzymol. 1995; 262: 613-627Crossref PubMed Scopus (204) Google Scholar). Briefly, the first-dimension gel was a 0.4% agarose gel run in 0.5× TBE buffer for 30 h at 0.75 V/cm at room temperature. The desired gel lane was sliced from the first-dimension gel and cast across the top of the second-dimension 1% agarose gel, which was run in 0.5× TBE buffer containing 0.3 μg/ml ethidium bromide for 16 h at 4.5 V/cm at 4 °C. For Southern hybridization, agarose gels were transferred to a Nytran Super-Charge nylon transfer membrane (Schleicher & Schuell Bioscience, Inc) by the downward sponge method (30Ming Y.Z. Di X. Gomez-Sanchez E.P. Gomez-Sanchez C.E. BioTechniques. 1994; 16: 58-59PubMed Google Scholar). The probe for the Southern blots consisted of a PCR fragment containing T4 origin ori(34) (T4 map positions 149.172-152.033 kb) labeled with [α-32P]dATP by using the Random Primed DNA Labeling kit (Roche Applied Sciences). Cloning and Expression of UvsW—UvsW and UvsW-K141R had been cloned and expressed as GST fusion proteins previously by this laboratory (15Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (68) Google Scholar). The purified GST-UvsW wild-type fusion protein possesses a branched-DNA specific and ATP-dependent helicase activity, and the lysine to arginine substitution (in the Walker A motif) abolished both ATPase and helicase activities. We sought untagged versions of the proteins for further biochemical characterization. To this end we cloned UvsW and the UvsW-K141R mutant into a GST fusion expression vector containing a PreScission protease (Amersham Biosciences) recognition site in the GST linker region. PreScission protease also has a GST fusion tag, allowing it to be used on-column to cleave glutathione-bound fusion proteins containing the recognition site. The cleaved protein product is then eluted without the need for glutathione, leaving the protease bound to the column. This strategy worked well for generating substantial amounts of the highly purified UvsW-K141R mutant protein (Fig. 1, lane 1). Attempts to obtain the wild-type UvsW protein using this system, however, were hampered by plasmid instability and low copy number. Because UvsW is known to unwind R-loops at replication initiation sites, we reasoned that replacing the R-loop-dependent ColE1-based plasmid origin with an R6K γ-based replication origin might improve UvsW production. The R6K γ origin uses the π initiator protein with no R-loop intermediate (31Abhyankar M.M. Reddy J.M. Sharma R. Bullesbach E. Bastia D. J. Biol. Chem. 2004; 279: 6711-6719Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The plasmid was found to be stable in strain BW23322, which encodes a mutated version of π (pir-116) that results in an increased plasmid copy number (32Metcalf W.W. Jiang W. Wanner B.L. Gene (Amst.). 1994; 138: 1-7Crossref PubMed Scopus (151) Google Scholar). Initially, we found that UvsW was highly contaminated with RNA and a co-purifying protein which was identified by mass spectral analysis to be the chaperonin GroEL. Both contaminants were effectively eliminated by including an RNase treatment and an incubation with ATP/Mg2+ during the purification process (33Thain A. Gaston K. Jenkins O. Clarke A.R. Trends Genet. 1996; 12: 209-210Abstract Full Text PDF PubMed Scopus (51) Google Scholar). Using these techniques and additional modifications of the standard extraction procedure, we were eventually able to obtain ∼600 μg of highly purified, soluble UvsW protein from 1 liter of culture (Fig. 1, lane 2). Unwinding of Oligonucleotide Substrates—The GST-UvsW fusion protein was previously shown to unwind a blunt-ended, fully duplex, branched-DNA Y substrate in the presence of ATP (15Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (68) Google Scholar). We began by analyzing the unwinding activity in more detail using the oligonucleotide-based substrates shown in Fig. 2. As in previous studies (15Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (68) Google Scholar), UvsW was able to unwind a duplex DNA Y substrate, but not a blunt-ended linear DNA substrate (Fig. 2). We did not detect significant unwinding of double-stranded substrates with either 5′ or 3′ single-stranded overhangs (the latter contrary to an earlier report (18Nelson S.W. Benkovic S.J. J. Biol. Chem. 2007; 282: 407-416Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar)), or double-stranded DNA with a flayed single-stranded end (Fig. 2). In contrast, UvsW efficiently unwound the static X-junction into flayed duplexes (Fig. 2B). At an enzyme/substrate rati
Referência(s)