Sequence Specificity and Biochemical Characterization of the RusA Holliday Junction Resolvase of Escherichia coli
1997; Elsevier BV; Volume: 272; Issue: 23 Linguagem: Inglês
10.1074/jbc.272.23.14873
ISSN1083-351X
AutoresSau N. Chan, Lynda K. Harris, Edward L. Bolt, Matthew C. Whitby, Robert G. Lloyd,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoThe RusA protein of Escherichia coliis an endonuclease that resolves Holliday intermediates in recombination and DNA repair. Analysis of its subunit structure revealed that the native protein is a dimer. Its resolution activity was investigated using synthetic X-junctions with homologous cores. Resolution occurs by dual strand incision predominantly 5′ of CC dinucleotides located symmetrically. A junction lacking homology is not resolved. The efficiency of resolution is related inversely to the number of base pairs in the homologous core, which suggests that branch migration is rate-limiting. Inhibition of resolution at high ratios of protein to DNA suggests that binding of RusA may immobilize the junction point at non-cleavable sites. Resolution is stimulated by alkaline pH and by Mn2+. The protein is unstable in the absence of substrate DNA and loses ∼80% of its activity within 1 min under standard reaction conditions. DNA binding stabilizes the activity. Junction resolution is inhibited in the presence of RuvA. This observation probably explains why RusA is unable to promote efficient recombination and DNA repair in ruvA+strains unless it is expressed at a high level. The RusA protein of Escherichia coliis an endonuclease that resolves Holliday intermediates in recombination and DNA repair. Analysis of its subunit structure revealed that the native protein is a dimer. Its resolution activity was investigated using synthetic X-junctions with homologous cores. Resolution occurs by dual strand incision predominantly 5′ of CC dinucleotides located symmetrically. A junction lacking homology is not resolved. The efficiency of resolution is related inversely to the number of base pairs in the homologous core, which suggests that branch migration is rate-limiting. Inhibition of resolution at high ratios of protein to DNA suggests that binding of RusA may immobilize the junction point at non-cleavable sites. Resolution is stimulated by alkaline pH and by Mn2+. The protein is unstable in the absence of substrate DNA and loses ∼80% of its activity within 1 min under standard reaction conditions. DNA binding stabilizes the activity. Junction resolution is inhibited in the presence of RuvA. This observation probably explains why RusA is unable to promote efficient recombination and DNA repair in ruvA+strains unless it is expressed at a high level. The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates made during homologous genetic recombination and DNA repair (1Sharples G.J. Chan S.C. Mahdi A.A. Whitby M.C. Lloyd R.G. EMBO J. 1994; 13: 6133-6142Google Scholar). Proteins of this type were first described in bacteriophage-infected bacterial cells (2Mizuuchi K. Kemper B. Hays J. Weisberg R. Cell. 1982; 29: 357-365Google Scholar, 3Kemper B. Garabett M. Eur. J. Biochem. 1981; 115: 123-131Google Scholar, 4de Massey B. Weisberg R.A. Studier F.W. J. Mol. Biol. 1987; 193: 359-376Google Scholar, 5de Massey B. Studier F.W. Dorgai L. Appelbaum F. Weisberg R.A. Cold Spring Harbor Symp. Quant. Biol. 1984; 49: 715-726Google Scholar), but have since been found in bacteria (6Iwasaki H. Takahagi M. Shida T. Nakata A. Shinagawa H. EMBO J. 1991; 10: 4381-4389Google Scholar, 7Connolly B. Parsons C. Benson F.E. Dunderdale H.J. Sharples G.J. Lloyd R.G. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6063-6067Google Scholar), yeast (8Symington L.S. Kolodner R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7247-7251Google Scholar, 9West S.C. Körner A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6445-6449Google Scholar, 10West S.C. Parsons C.A. Picksley S.M. J. Biol. Chem. 1987; 262: 12752-12758Google Scholar), and mammalian cells (11Elborough K.M. West S.C. EMBO J. 1990; 9: 2931-2936Google Scholar, 12Hyde H. Davies A. Benson F.E. West S.C. J. Biol. Chem. 1994; 269: 5202-5209Google Scholar). The T4 enzyme, endonuclease VII, cleaves a variety of DNA secondary structures including Holliday junctions (2Mizuuchi K. Kemper B. Hays J. Weisberg R. Cell. 1982; 29: 357-365Google Scholar), cruciforms (13Lilley D.M.J. Kemper B. Cell. 1984; 36: 413-422Google Scholar), branched structures (14Jensch F. Kemper B. EMBO J. 1986; 5: 181-189Google Scholar, 15Duckett D.R. Murchie A.H. Diekmann S. Kitzing E.V. Kemper B. Lilley D.M. Cell. 1988; 55: 79-89Google Scholar), heteroduplex loops (16Kleff S. Kemper B. EMBO J. 1988; 7: 1527-1535Google Scholar), and mismatches (17Solaro P.C. Birkenkamp K. Pfeiffer P. Kemper B. J. Mol. Biol. 1993; 230: 868-877Google Scholar). This broad substrate spectrum is consistent with a role both in recombination and in the removal of branch structures from the phage DNA prior to packaging (18Kemper B. Pottmeyer S. Solaro P. Kosak H.G. Sarma R.H. Sarma M.H. Structure and Methods: Human Genome Initiative and DNA Recombination. Adenine Press, New York1990: 215-229Google Scholar). RuvC protein of E. coli is highly selective for Holliday junctions (19Dunderdale H.J. Benson F.E. Parsons C.A. Sharples G.J. Lloyd R.G. West S.C. Nature. 1991; 354: 506-510Google Scholar, 20Benson F.E. West S.C. J. Biol. Chem. 1994; 269: 5195-5201Google Scholar, 21Takahagi M. Iwasaki H. Shinagawa H. J. Biol. Chem. 1994; 269: 15132-15139Google Scholar), which suggests a more specialized role in recombination. It binds the four-way duplex structure of a Holliday junction and folds the DNA in an open configuration that allows symmetrical strands to be located within the catalytic core of each subunit and cleaved to yield nicked duplex products (22Bennett R.J. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5635-5639Google Scholar, 23Bennett R.J. West S.C. J. Mol. Biol. 1995; 252: 213-226Google Scholar, 24Bennett R.J. West S.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12217-12222Google Scholar). Strand cleavage has been shown to involve four acidic residues in the protein (25Ariyoshi M. Vassylyev D.G. Iwasaki H. Nakamura H. Shinagawa H. Morikawa K. Cell. 1994; 78: 1063-1072Google Scholar, 26Saito A. Iwasaki H. Ariyoshi M. Morikawa K. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7470-7474Google Scholar) and occurs 5′ of a T that has to be located within a certain sequence context for efficient resolution (27Shah R. Bennet R.J. West S.C. Cell. 1994; 79: 853-864Google Scholar, 28Shida T. Iwasaki H. Saito A. Kyogoku Y. Shinagawa H. J. Biol. Chem. 1996; 271: 26105-26109Google Scholar). This sequence-dependence reflects both topological factors that dictate folding of the junction (28Shida T. Iwasaki H. Saito A. Kyogoku Y. Shinagawa H. J. Biol. Chem. 1996; 271: 26105-26109Google Scholar) and the need to make specific protein-DNA contacts (29Shah R. Cosstick R. West S.C. EMBO J. 1997; 16: 1464-1472Google Scholar). RusA was discovered through its ability to promote recombination and DNA repair in strains lacking RuvC. Genetic analysis of this alternative resolvase provided the first indication that RuvC cannot cleave junctions efficiently in vivo without the associated activities of RuvA and RuvB (1Sharples G.J. Chan S.C. Mahdi A.A. Whitby M.C. Lloyd R.G. EMBO J. 1994; 13: 6133-6142Google Scholar, 30Mandal T.N. Mahdi A.A. Sharples G.J. Lloyd R.G. J. Bacteriol. 1993; 175: 4325-4334Google Scholar). These two proteins assemble at junctions to form a highly specialized RuvAB complex that drives the branch point along the DNA (31Parsons C.A. Stasiak A. Bennett R.J. West S.C. Nature. 1995; 374: 375-378Google Scholar, 32Stasiak A. Tsaneva I.R. West S.C. Benson C.J.B. Yu X. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7618-7622Google Scholar, 33West S.C. J. Bacteriol. 1996; 178: 1237-1241Google Scholar, 34Rafferty J.B. Sedelnikova S.E. Hargreaves D. Artymiuk P.J. Baker P.J. Sharples G.J. Mahdi A.A. Lloyd R.G. Rice D.W. Science. 1996; 274: 415-421Google Scholar). They presumably help RuvC to target cleavable sequences by holding the junction in an open configuration (RuvA) and moving it along the DNA (RuvB), possibly through formation of a RuvABC "resolvasome" complex (34Rafferty J.B. Sedelnikova S.E. Hargreaves D. Artymiuk P.J. Baker P.J. Sharples G.J. Mahdi A.A. Lloyd R.G. Rice D.W. Science. 1996; 274: 415-421Google Scholar, 35Whitby M.C. Bolt E.L. Chan S.N. Lloyd R.G. J. Mol. Biol. 1996; 264: 878-890Google Scholar). RusA has no requirement for RuvAB. However, efficient recombination and DNA repair does need RecG which like RuvAB drives branch migration of Holliday junctions (30Mandal T.N. Mahdi A.A. Sharples G.J. Lloyd R.G. J. Bacteriol. 1993; 175: 4325-4334Google Scholar, 36Mahdi A.A. Sharples G.J. Mandal T.N. Lloyd R.G. J. Mol. Biol. 1996; 257: 561-573Google Scholar, 37Lloyd R.G. Sharples G.J. EMBO J. 1993; 12: 17-22Google Scholar, 38Whitby M.C. Ryder L. Lloyd R.G. Cell. 1993; 75: 341-350Google Scholar). Whether or not this reflects a need to locate junctions at cleavable sequences remains to be determined. The 14-kDa RusA polypeptide is encoded by a gene (rusA) located within the defective prophage, DLP12, and is probably of bacteriophage origin (36Mahdi A.A. Sharples G.J. Mandal T.N. Lloyd R.G. J. Mol. Biol. 1996; 257: 561-573Google Scholar). The rusA gene is normally expressed poorly, if at all, and can be deleted with no apparent effect. However, it can be activated to suppress the ruvmutant phenotype by insertion of either IS2 or IS10 (formerly called rus-1 and rus-2mutations, respectively) upstream of the gene to promote transcription, or by cloning in a multicopy plasmid (1Sharples G.J. Chan S.C. Mahdi A.A. Whitby M.C. Lloyd R.G. EMBO J. 1994; 13: 6133-6142Google Scholar, 30Mandal T.N. Mahdi A.A. Sharples G.J. Lloyd R.G. J. Bacteriol. 1993; 175: 4325-4334Google Scholar, 36Mahdi A.A. Sharples G.J. Mandal T.N. Lloyd R.G. J. Mol. Biol. 1996; 257: 561-573Google Scholar). Expression from multicopy plasmids is needed to achieve full suppression in the presence of RuvA, which suggests that the binding of Holliday junctions by RuvA can prevent their resolution by RusA (1Sharples G.J. Chan S.C. Mahdi A.A. Whitby M.C. Lloyd R.G. EMBO J. 1994; 13: 6133-6142Google Scholar, 30Mandal T.N. Mahdi A.A. Sharples G.J. Lloyd R.G. J. Bacteriol. 1993; 175: 4325-4334Google Scholar). In this work, we detail a new purification of RusA and investigate the native form of the protein. We also report its general biochemical properties, its sequence specificity, and the inhibition of its resolution activity by RuvA. E. coli strain N3757 (1Sharples G.J. Chan S.C. Mahdi A.A. Whitby M.C. Lloyd R.G. EMBO J. 1994; 13: 6133-6142Google Scholar) is a ΔruvAC65 eda-51::Tn10derivative of BL21 (DE3) plysS (39Studier F.W. Rosenberg A.H. Dunn J.J. Methods Enzymol. 1991; 185: 60-89Google Scholar). pAM151 (1Sharples G.J. Chan S.C. Mahdi A.A. Whitby M.C. Lloyd R.G. EMBO J. 1994; 13: 6133-6142Google Scholar) is a derivative of pT7-7 carrying rusA+ under control of the phage T7 φ10 promoter (39Studier F.W. Rosenberg A.H. Dunn J.J. Methods Enzymol. 1991; 185: 60-89Google Scholar). RuvA (40Tsaneva I.R. Illing G.T. Lloyd R.G. West S.C. Mol. Gen. Genet. 1992; 235: 1-10Google Scholar) and RuvC (41Dunderdale H.J. Sharples G.J. Lloyd R.G. West S.C. J. Biol. Chem. 1994; 269: 5187-5194Google Scholar) were purified as described elsewhere. T4 polynucleotide kinase was from Pharmacia Biotech Inc., and [γ-32P]ATP from Amersham. All other reagents were from Sigma or BDH and were of analytical grade. Heparin-agarose, Reactive Blue 4-agarose, and double-stranded DNA cellulose were purchased from Sigma. Phosphocellulose P11 was from Whatman, and DEAE Bio-Gel A from Bio-Rad. A pre-packed gel filtration column (Superose 12 HR 10/30) was from Pharmacia. All chromatography was carried out at 4 °C in buffer A (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 0.5 mm DTT, 1The abbreviations used are:DTTdithiothreitolMES4-morpholineethanesulfonic acidMOPS4-morpholinepropanesulfonic acidPAGEpolyacrylamide gel electrophoresis 10% glycerol). Phenylmethylsufonyl fluoride and KCl or NaCl were added to buffer A as indicated. dithiothreitol 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid polyacrylamide gel electrophoresis SDS-PAGE analysis of protein samples was conducted using 15% gels and followed standard procedures (42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Unless stated otherwise, samples were mixed with SDS loading buffer (100 mm Tris-HCl, pH 6.8, 200 mm DTT, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and boiled for 5 min before electrophoresis. Molecular mass markers (Bio-Rad) were rabbit muscle phosphorylase b (97,000), bovine serum albumin (67,000), hen egg white albumin (45,000), bovine carbonic anhydrase (31,000), soybean trypsin inhibitor (21Takahagi M. Iwasaki H. Shinagawa H. J. Biol. Chem. 1994; 269: 15132-15139Google Scholar, 500), and hen egg white lysozyme (14,400). Gels were stained with Coomassie Brilliant Blue, or silver-stained as described (43Morrissey J.H. Anal. Biochem. 1981; 117: 307-310Google Scholar). 500-ml batches of strain N3757 transformed with pAM151 were grown with aeration at 37 °C in Luria-Bertani broth containing 10 g/liter NaCl, 100 μg/ml ampicillin, and 20 μg/ml chloramphenicol. At a cell density corresponding to anA650 of 0.5, RusA was induced by adding isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 2 mm and incubating for a further 3 h. The cells were chilled on ice, harvested by centrifugation, resuspended in 10 ml of lysis buffer (100 mm Tris-HCl, pH 8.0, 2 mm EDTA, 5% glycerol), and stored at −80 °C until required. Under these conditions, RusA was induced to 15–30% of the total cell protein. Frozen cells (70 ml) from 5 liters of induced culture were thawed at room temperature and then mixed on ice with 25 ml of 5 m NaCl, 0.5 ml 200 mm DTT, 1 ml of 10% (v/v) Triton X-100. Preliminary trials revealed that a high salt concentration was required during cell lysis to reduce loss of RusA. Cells were lysed by freezing in liquid nitrogen and rethawing, and cell debris was removed by centrifugation at 40,000 rpm for 60 min at 4 °C, using a Kontron TST 41.14 rotor. Despite the presence of high salt, ∼70% of the RusA was removed with the cell debris. All subsequent steps were carried out at 4 °C. Purification of RusA was monitored throughout by SDS-PAGE analysis of column fractions and by assays for junction cleavage activity, using J12 DNA as a substrate. The cleavage assays also provided a measure of nonspecific nuclease contamination. The supernatant (85 ml, 978 mg of protein) fraction containing the soluble RusA was dialyzed overnight against 4 liters of buffer A containing 1 mm phenylmethylsulfonyl fluoride and 0.5 m KCl, centrifuged at 15,000 rpm for 10 min to remove traces of insoluble material, and applied to a phosphocellulose column (2.6 × 8.5 cm, 45-ml bed volume) equilibrated in the same buffer. The column was washed with 100 ml of the same buffer before eluting bound proteins with a 450 ml of gradient of 0.5–1.0 m KCl in buffer A. RusA eluted between 0.7 and 0.9 m KCl. Peak fractions were pooled and dialyzed against 2 liters of buffer A containing 0.1 m KCl. SDS-PAGE analysis revealed that RusA was >90% pure at this stage. Minor protein bands and trace exonuclease contaminants were removed by fractionation on three further columns. The dialyzed phosphocellulose pool (144 ml, 47 mg of protein) was loaded on a DEAE Bio-Gel A column (2.6 × 4.5 cm, 24-ml bed volume) and washed with 50 ml of the same buffer. Bio-Gel A does not bind RusA under these conditions, but retains contaminants that do not fractionate from RusA in later chromatographic steps. The flow-through (∼200 ml, 32 mg of protein) was applied in aliquots of 50 ml to a double stranded DNA-cellulose column (1.0 × 5.1 cm, 4-ml bed volume), washed with 8 ml of buffer A containing 0.25 mKCl, and bound proteins eluted with a 40-ml gradient of 0.25–1.0m KCl in buffer A. RusA eluted between 0.5 and 0.7m KCl. Peak fractions from the four aliquots were pooled (80 ml, 28 mg of protein), dialyzed against 2 liters of buffer A, and applied to a heparin-agarose column (1.0 × 7.6 cm, 6-ml bed volume). The column was washed with 12 ml of buffer A containing 0.25m KCl and bound proteins eluted with a 60-ml gradient of 0.25–1.0 m KCl in buffer A. RusA eluted between 0.5 and 0.7 m KCl. No contaminating bands could be detected in these fractions with Coomassie Blue or silver-staining of SDS gels. The peak fractions were pooled and dialyzed into storage buffer (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 0.5 mm DTT, 50% (v/v) glycerol), and stored in aliquots at −80 °C. All protein concentrations were determined by a modified Bradford method, using a protein assay kit from (Bio-Rad) and bovine serum albumin (Pharmacia) as standard. Amounts of RuvA, RuvC, and RusA are expressed as moles of the monomeric protein. A 200-μl sample of RusA (1 mg/ml protein) was dialyzed into TEA buffer (20 mm TEA-HCl, pH 8.5, 1 mm EDTA, 150 mm NaCl, 0.5 mm DTT, 10% glycerol), mixed with fresh glutaraldehyde (final concentration 14 mm), and incubated for 1 min at room temperature before storing at −20 °C. RusA (500 μg) was dialyzed for 5 h against buffer A containing KCl at either 0.15 or 1 m and a 300-μl sample applied to a precalibrated gel filtration column. Molecular mass standards (Bio-Rad) were bovine thyroglobulin (670,000), bovine γ-globulin (158,100), chicken ovalbumin (44,000), horse myoglobin (17,000), and cyanocobalamin (1,350). Protein was detected by measuring the absorbance at 280 nm. Oligonucleotides were synthesized on an Applied Biosystems 380B DNA synthesizer using cyanoethyl chemistry. Each oligonucleotide was deprotected, precipitated in ethanol, and purified on a 12% (w/v) polyacrylamide gel containing 7 murea. The bands containing full-length oligonucleotides were cut out and extracted from the gel by soaking in water overnight. X-junctions were made by annealing four partially complementary oligonucleotides, each of approximately 50 nucleotides in length. The point of strand crossover is either fixed centrally within the structure (static junction, J0) or free to branch migrate within a central core of homology (mobile junctions J2, J3, J4, J11, J12, and J26). The number after the letter J indicates the size of the homologous core in base pairs. The sequences of the oligonucleotides used for J0 (35Whitby M.C. Bolt E.L. Chan S.N. Lloyd R.G. J. Mol. Biol. 1996; 264: 878-890Google Scholar), J2 (26Saito A. Iwasaki H. Ariyoshi M. Morikawa K. Shinagawa H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7470-7474Google Scholar), J3 (35Whitby M.C. Bolt E.L. Chan S.N. Lloyd R.G. J. Mol. Biol. 1996; 264: 878-890Google Scholar), J4 (35Whitby M.C. Bolt E.L. Chan S.N. Lloyd R.G. J. Mol. Biol. 1996; 264: 878-890Google Scholar), J11 (27Shah R. Bennet R.J. West S.C. Cell. 1994; 79: 853-864Google Scholar, 35Whitby M.C. Bolt E.L. Chan S.N. Lloyd R.G. J. Mol. Biol. 1996; 264: 878-890Google Scholar), J12 (37Lloyd R.G. Sharples G.J. EMBO J. 1993; 12: 17-22Google Scholar, 44Parsons C.A. West S.C. J. Mol. Biol. 1993; 232: 397-405Google Scholar), and J26 (45Picksley S. Parsons C. Kemper B. West S. J. Mol. Biol. 1990; 212: 723-735Google Scholar) have been described. J4 was made by annealing oligonucleotides 5, 7, 9, and 10 (35Whitby M.C. Bolt E.L. Chan S.N. Lloyd R.G. J. Mol. Biol. 1996; 264: 878-890Google Scholar). J0 has been referred to elsewhere as X-static (35Whitby M.C. Bolt E.L. Chan S.N. Lloyd R.G. J. Mol. Biol. 1996; 264: 878-890Google Scholar) and J11 as junction A (27Shah R. Bennet R.J. West S.C. Cell. 1994; 79: 853-864Google Scholar). Annealing followed the procedures described (46Parsons C.A. Kemper B. West S.C. J. Biol. Chem. 1990; 265: 9285-9289Google Scholar). One of the strands was labeled at the 5′ end prior to annealing using [γ-32P]ATP and T4 polynucleotide kinase. Junctions were purified by nondenaturing 10% PAGE and electroelution. Unlabeled junctions were made as described above, except that oligonucleotides were annealed in equimolar ratios and the resulting substrates were not purified by PAGE. Unlabeled linear duplex DNA was made in the same way using oligonucleotides 1 and 5 described previously (37Lloyd R.G. Sharples G.J. EMBO J. 1993; 12: 17-22Google Scholar). Cleavage of32P-labeled junction DNA by RusA and RuvC was assayed at 37 °C in buffer CB (25 mm Tris-HCl, pH 8.0, 1 mm DTT, 100 μg/ml bovine serum albumin, 6% (v/v) glycerol) or SCB (25 mm Tris-HCl, pH 8.0, 1 mmDTT, 100 μg/ml bovine serum albumin, 10% (v/v) glycerol), with either MgCl2 or MnCl2 added as indicated (10 mm in standard resolution reactions). Reactions (20 μl final volume) were terminated by adding 5 μl of stop mixture (2.5% SDS, 200 mm EDTA, 10 mg/ml proteinase K) and incubating for a further 10 min at 37 °C to deproteinize the mixture. DNA products were analyzed by native PAGE, using 10% gels in TBE (90 mmTris borate, pH 8.0, 2 mm EDTA). Gels were dried, and labeled products were detected using a Molecular Dynamics PhosphorImager (Model 425) and by autoradiography. Reactions were quantified using ImageQuant software (Molecular Dynamics) to analyze PhosphorImages. For time courses, 20-μl samples were removed at intervals from bulk reactions and processed as described. To assess activity over a wide range of pH, assays were conducted in a series of different buffers, each covering a narrow range of pH, with a pH overlap between each buffer type. Separate preparations of junction DNA, each 5′-32P-labeled in a different strand, were incubated with RusA in SCB buffer for 10 min at 37 °C. Deproteinized samples were extracted with phenol, and the DNA was precipitated with ethanol, resuspended in sequencing gel loading dye (0.3% (w/v) bromphenol blue, 0.3% (w/v) xylene cyanol, 10 mm EDTA, pH 7.5, 97.5% formamide), and denatured by boiling for 5 min before analyzing by denaturing PAGE, using 12% gels (42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Sequencing ladders of each labeled oligonucleotide generated using a Maxam-Gilbert sequencing kit (Sigma) were loaded on the same gel to provide markers. Gels were dried, and analyzed by autoradiography and PhosphorImaging. Cleavage sites were mapped by reference to the sequencing ladder. A 1.5-base allowance was made to compensate for the nucleoside eliminated in the sequencing reaction. 32P-Labeled junction DNA (0.2–0.6 ng) was mixed with RusA, RuvA, or RuvC, in binding buffer (50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 1 mm DTT, 100 μg/ml bovine serum albumin, 6% (v/v) glycerol) in a final volume of 20 μl. After 10–15 min on ice, protein-DNA complexes were resolved by nondenaturing PAGE using 4% gels in low ionic strength buffer (6.7 mm Tris-HCl, pH 8.0, 3.3 mm sodium acetate, 2 mm EDTA). Gels were cooled to 4 °C before use, but electrophoresis was at room temperature with continuous buffer recirculation. Gels were dried and analyzed by autoradiography. In previous work, we described a recombinant plasmid (pAM151) for the overexpression of RusA and purified some of the protein from strain N3757 transformed with this construct (1Sharples G.J. Chan S.C. Mahdi A.A. Whitby M.C. Lloyd R.G. EMBO J. 1994; 13: 6133-6142Google Scholar). To obtain larger quantities of the protein for physical analyses, we devised a new purification ("Materials and Methods"). This yielded 24 mg of RusA (in 21 ml) from 5 liters of induced cells. The recovery was much lower than expected given that RusA accounted for 15–20% of the total cell protein. A large fraction of the induced protein was in an insoluble form that was removed with the cell debris during centrifugation of the lysed cells. Subsequent studies revealed that solubility can be improved by growing cells and inducing RusA at 25 °C (data not shown), allowing much better recovery of the induced protein by the same method. The protein purified was free of any visible contaminants as judged by Coomassie Blue and silver staining of SDS gels (Fig. 1A, lane c, and data not shown). To investigate the native form of RusA, 11 μg of the protein was analyzed by SDS-PAGE with and without prior boiling in SDS sample loading buffer. The boiled sample showed a single band of RusA (Fig.1A, lane c). It migrates as a 15-kDa species, which is close to its predicted molecular mass of 13.8 kDa (1Sharples G.J. Chan S.C. Mahdi A.A. Whitby M.C. Lloyd R.G. EMBO J. 1994; 13: 6133-6142Google Scholar). Without boiling, a second band is clearly visible. Its migration suggests a molecular mass of about 30 kDa, which is consistent with a dimer of RusA. A further sample of the protein was treated with glutaraldehyde before boiling in sample loading buffer containing SDS. Fig. 1B shows a 30-kDa protein species in the treated sample (lane b). The only other band visible corresponds to the monomer species seen in the untreated control (lane c). The absence of any other band suggests that glutaraldehyde specifically cross-links a dimer of RusA. The ability to detect a dimer on denaturing SDS gels without prior cross-linking (Fig. 1A, lane b) suggests that a substantial fraction of the purified protein is in this form. To determine the molecular mass, a sample of the native protein was dialyzed against buffer A containing 1 m KCl and applied to a Superose 12 HR column. The elution profile (Fig. 1C) showed a single sharp peak. SDS-PAGE analysis of the fractions confirmed that this peak coincided with the peak of RusA (data not shown). By comparison with protein standards analyzed using the same buffer, the molecular mass of the protein at the peak was determined to be 24 kDa. This is nearly twice the value of 13.8 kDa predicted from the amino acid sequence of the protein (36Mahdi A.A. Sharples G.J. Mandal T.N. Lloyd R.G. J. Mol. Biol. 1996; 257: 561-573Google Scholar). Given the high salt conditions, this result strongly supports the idea that RusA is a dimer in solution. We also analyzed RusA in the presence of 0.15 m KCl. A substantial fraction of the protein eluted faster than the 670-kDa marker, suggesting a tendency to form aggregates. Previous studies (1Sharples G.J. Chan S.C. Mahdi A.A. Whitby M.C. Lloyd R.G. EMBO J. 1994; 13: 6133-6142Google Scholar) showed that RusA resolves X-junctions to nicked duplex products. Resolution can be monitored by electrophoresis of the reaction products on a nondenaturing polyacrylamide gel (Fig.2A). Using this simple assay, we investigated the resolution activity of RusA over a range of protein concentrations in reaction buffer containing 10 mm MgCl2 or MnCl2. Fig. 2B shows the result of a typical experiment. Resolution is clearly sensitive to the concentration of RusA, peaking sharply at 50 nm in the Mn2+reactions and at 200 nm in the Mg2+ reactions. Above these levels, the activity of RusA is very clearly inhibited. At subinhibitory concentrations, RusA is substantially more active in Mn2+ than it is in Mg2+ (Fig. 2B, inset). This difference disappears as RusA becomes inhibitory. Furthermore, even with 5 μm RusA in the reaction, resolution is not totally abolished. Similar results were obtained using junction J3 (data not shown), although the fraction of junction resolved was generally higher (see below). Autoinhibition in this case reached a lower limit of ∼30% junction resolution (data not shown). To determine the optimal conditions for resolution by RusA, we investigated the activity under a variety of reaction conditions, using J12 with RusA at 100 nm. RusA showed a requirement for divalent metal ions with a broad optimum of 10–30 mm for Mg2+ (Fig. 3A). Replacing Mg2+ with Mn2+ stimulated resolution in the 2–10 mm range, but at 20 mm and above Mn2+ was substantially less effective than Mg2+(data not shown). No activity was detected with Zn2+, Ca2+, or Cu2+. RusA was insensitive to NaCl up to 200 mm, but was inhibited at higher concentrations and inactive at 500 mm (Fig. 3B). Activity in Mg2+ buffer was stimulated at alkaline pH, and increased with increasing pH throughout the range tested (Fig.4A). In contrast, the activity in Mn2+ buffer was optimal at pH 7.5. Up to pH 8.5, it was also significantly higher than in Mg2+ buffer. Fig.4B shows the time course of the reaction in the presence of 10 mm Mg2+ under two different buffer conditions, one at pH 7.5 in Tris buffer, and one at pH 9.5 in glycine buffer. As expected, significantly more of the junction was resolved at the higher pH. In both cases the reaction was over within the first 10–15 min even though substantial fractions of the substrate remained unresolved. The failure to cleave all the junction molecules is surprising given that the protein (in terms of dimers) was present in at least a 50-fold molar excess. It means either RusA is inactivated rapidly during the reaction or that a fraction of the junction DNA, determined by the pH, is resistant to cleavage. It could also reflect a combination of these factors.Figure 4Effect of pH on resolution activity of RusA.A, resolution activity was measured over a pH range in the presence of 10 mm MgCl2 or 10 mm MnCl2 using the following buffers, all at 40 mm and containing 1 mm DTT, 100 μg/ml bovine serum albumin:sodium acetate, pH 5.0 and 5.5; MES, pH 6.0 and 6.5; MOPS, pH 7.0; Tris-HCl, pH 7.5, 8.0, and 8.5; glycine, pH 9.0 and 9.5. Reactions contained 0.3 ng of 32P-labeled J12 DNA and 100 nm RusA, and were incubated for 10 min at 37 °C before processing as described. Values are means of 2 or 3 assays at each pH.Error bars show the standard errors of the mean values.B, time course of J12 resolution at pH 7.5 and 9.5. Bulk reaction mixtures (160 μl) containing 2.4 ng of32P-labeled J12 DNA, and RusA at a final concentration of 100 nm, in buffer SCB, pH 7.5, or SCB with 40 mm glycine, pH 9.5, replacing Tris-HCl, pH 7.5, were incubated at 37 °C. Samples (20 μl) were withdrawn at intervals and processed as described. The results at
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