Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Mutation of a Highly Conserved Arginine in Motif IV ofEscherichia coli DNA Helicase II Results in an ATP-binding Defect

1997; Elsevier BV; Volume: 272; Issue: 30 Linguagem: Inglês

10.1074/jbc.272.30.18614

ISSN

1083-351X

Autores

Mark C. Hall, Steven W. Matson,

Tópico(s)

Enterobacteriaceae and Cronobacter Research

Resumo

A site-directed mutation in motif IV ofEscherichia coli DNA helicase II (UvrD) was generated to examine the functional significance of this region. The highly conserved arginine at position 284 was replaced with alanine to construct UvrD-R284A. The ability of the mutant allele to function in methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair was examined by genetic complementation assays. The R284A substitution abolished function in both DNA repair pathways. To identify the biochemical defects responsible for the loss of biological function, UvrD-R284A was purified to apparent homogeneity, and its biochemical properties were compared with wild-type UvrD. UvrD-R284A failed to unwind a 92-base pair duplex region and was severely compromised in unwinding a 20-base pair duplex region. TheK m of UvrD-R284A for ATP was significantly greater than 3 mm compared with 80 μm for UvrD. A large decrease in ATP binding was confirmed using a nitrocellulose filter binding assay. These data suggested that the R284A mutation severely reduced the affinity of helicase II for ATP. The reduced unwinding activity and loss of biological function of UvrD-R284A was probably the result of decreased affinity for ATP. These results implicate motif IV of superfamily I helicases in nucleotide binding and represent the first characterization of a helicase mutation outside motifs I and II that severely impacted the K m for ATP. A site-directed mutation in motif IV ofEscherichia coli DNA helicase II (UvrD) was generated to examine the functional significance of this region. The highly conserved arginine at position 284 was replaced with alanine to construct UvrD-R284A. The ability of the mutant allele to function in methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair was examined by genetic complementation assays. The R284A substitution abolished function in both DNA repair pathways. To identify the biochemical defects responsible for the loss of biological function, UvrD-R284A was purified to apparent homogeneity, and its biochemical properties were compared with wild-type UvrD. UvrD-R284A failed to unwind a 92-base pair duplex region and was severely compromised in unwinding a 20-base pair duplex region. TheK m of UvrD-R284A for ATP was significantly greater than 3 mm compared with 80 μm for UvrD. A large decrease in ATP binding was confirmed using a nitrocellulose filter binding assay. These data suggested that the R284A mutation severely reduced the affinity of helicase II for ATP. The reduced unwinding activity and loss of biological function of UvrD-R284A was probably the result of decreased affinity for ATP. These results implicate motif IV of superfamily I helicases in nucleotide binding and represent the first characterization of a helicase mutation outside motifs I and II that severely impacted the K m for ATP. Helicase-catalyzed unwinding of double-stranded nucleic acid molecules is required in all aspects of DNA and RNA metabolism including replication, DNA repair, recombination, transcription, translation, RNA processing, and bacterial conjugation (1Ray B.K. Lawson T.G. Kramer J.C. Cladaras M.H. Grifo J.A. Abramson R.D. Merrick W.C. Thach R.E. J. Biol. Chem. 1985; 260: 7651-7658Abstract Full Text PDF PubMed Google Scholar, 2Schmid S.R. Linder P. Mol. Microbiol. 1992; 6: 283-292Crossref PubMed Scopus (449) Google Scholar, 3Wasserman D.A. Steitz J.A. Nature. 1991; 349: 463-464Crossref PubMed Scopus (180) Google Scholar, 4Brennan C.A. Dombroski A.J. Platt T. Cell. 1987; 48: 945-952Abstract Full Text PDF PubMed Scopus (185) Google Scholar, 5Lohman T.M. Mol. Microbiol. 1992; 6: 5-14Crossref PubMed Scopus (133) Google Scholar, 6Lohman T.M. J. Biol. Chem. 1993; 268: 2269-2272Abstract Full Text PDF PubMed Google Scholar, 7Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (671) Google Scholar, 8Matson S.W. Kaiser-Rogers K.A. Annu. Rev. Biochem. 1990; 59: 289-329Crossref PubMed Scopus (336) Google Scholar, 9Matson S.W. Prog. Nucleic Acid Res. Mol. Biol. 1991; 40: 289-326Crossref PubMed Scopus (76) Google Scholar, 10Matson S.W. George J.W. Bean D.W. Bioessays. 1994; 16: 13-22Crossref PubMed Scopus (270) Google Scholar). Helicases couple the energy derived from hydrolysis of nucleoside 5′-triphosphates (NTPs) 1The abbreviations used are: NTP, nucleoside 5′-triphosphate; ssDNA, single-stranded DNA; LB, Luria broth; ATPγS, adenosine 5′-O-(thiotriphosphate); bp, base pair. 1The abbreviations used are: NTP, nucleoside 5′-triphosphate; ssDNA, single-stranded DNA; LB, Luria broth; ATPγS, adenosine 5′-O-(thiotriphosphate); bp, base pair. to the disruption of hydrogen bonds between the complementary bases of a double helix. The mechanism of unwinding is not known although models have been proposed and are currently being tested (7Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (671) Google Scholar, 11Wong I. Lohman T.M. Science. 1992; 256: 350-355Crossref PubMed Scopus (166) Google Scholar, 12Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar). These models are based on the formation of active oligomers, which provide multiple DNA binding sites for the helicase. Most, if not all, helicases appear capable of forming either a dimer or hexamer, and evidence suggests that the oligomer is an active species (7Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (671) Google Scholar). Helicases are ubiquitous in nature, with numerous examples in viral, prokaryotic, and eukaryotic organisms. Extensive computer-assisted sequence analysis of numerous helicases has uncovered a series of short, conserved amino acid motifs (13Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. FEBS Lett. 1988; 235: 16-24Crossref PubMed Scopus (211) Google Scholar, 14Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. Nucleic Acids Res. 1989; 17: 4713-4729Crossref PubMed Scopus (829) Google Scholar, 15Hodgman T.C. Nature. 1988; 333: 22-23Crossref PubMed Scopus (323) Google Scholar). This has allowed grouping of helicases into four families based on the extent of amino acid similarity and on the organization of these conserved regions. These families presumably represent evolutionary relationships. Superfamilies I and II are the largest and most closely related groups, whereas superfamily III and family IV have unique motif compositions that differ considerably from those in superfamilies I and II and from each other (16Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1032) Google Scholar). Analysis of helicases with mutations in highly conserved residues in several of the so-called “helicase motifs” has suggested a biochemical role of some of these regions in helicase function. For example, motifs I and II, first described as the Walker A and B sequences in a large family of NTP binding proteins (17Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4249) Google Scholar), have been directly implicated in NTP binding and/or hydrolysis (18George J.W. Robert M. Brosh J. Matson S.W. J. Mol. Biol. 1994; 235: 424-435Crossref PubMed Scopus (81) Google Scholar, 19Brosh Jr., R.M. Matson S.W. J. Bacteriol. 1995; 177: 5612-5621Crossref PubMed Google Scholar, 20Washburn B.K. Kushner S.R. J. Bacteriol. 1993; 175: 341-350Crossref PubMed Google Scholar, 21Pause A. Sonenberg N. EMBO J. 1992; 11: 2643-2654Crossref PubMed Scopus (530) Google Scholar, 22Zavitz K.H. Marians K.J. J. Biol. Chem. 1992; 267: 6933-6940Abstract Full Text PDF PubMed Google Scholar). The function of the remaining helicase motifs is less clear. Roles for motif VI in nucleic acid binding and ATP hydrolysis have been proposed for various superfamily II RNA helicases (23Fernandez A. Lain S. Garcia J.A. Nucleic Acids Res. 1995; 23: 1327-1332Crossref PubMed Scopus (73) Google Scholar, 24Gross C.H. Shuman S. J. Virol. 1996; 70: 1706-1713Crossref PubMed Google Scholar, 25Pause A. Methot N. Sonenberg N. Mol. Cell. Biol. 1993; 13: 6789-6798Crossref PubMed Scopus (259) Google Scholar). Motif V has been implicated in single-stranded DNA (ssDNA) binding (26Graves-Woodward K.L. Weller S.K. J. Biol. Chem. 1996; 271: 13629-13635Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and motif III in coordination of ATP and ssDNA binding (27Brosh Jr., R.M. Matson S.W. J. Biol. Chem. 1996; 271: 25360-25368Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) for superfamily I helicases. The recent crystal structure of PcrA, a superfamily I DNA helicase fromBacillus stearothermophilus, suggested that all seven of the conserved helicase motifs are clustered together in the vicinity of the ATP binding site (28Subramanya H.S. Bird L.E. Brannigan J.A. Wigley D.B. Nature. 1996; 384: 379-383Crossref PubMed Scopus (381) Google Scholar). Thus, all of the motifs may be involved in ATP binding and/or hydrolysis, at least for those enzymes with structures similar to PcrA. Escherichia coli DNA helicase II, the product of theuvrD gene, is a well characterized DNA helicase. This enzyme is a required component of the UvrABC-mediated nucleotide excision repair pathway (29Husain I. Van-Houten B. Thomas D.C. Abdel-Monem M. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6774-6778Crossref PubMed Scopus (146) Google Scholar, 30Caron P.R. Kushner S.R. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4925-4929Crossref PubMed Scopus (138) Google Scholar) and the methyl-directed mismatch repair pathway (31Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (446) Google Scholar). Less defined roles in recombination and DNA replication have also been suggested (18George J.W. Robert M. Brosh J. Matson S.W. J. Mol. Biol. 1994; 235: 424-435Crossref PubMed Scopus (81) Google Scholar, 19Brosh Jr., R.M. Matson S.W. J. Bacteriol. 1995; 177: 5612-5621Crossref PubMed Google Scholar, 32Horii Z.I. Clark A.J. J. Mol. Biol. 1973; 80: 327-344Crossref PubMed Scopus (310) Google Scholar, 33Mendonca V.M. Kaiser-Rogers K. Matson S.W. J. Bacteriol. 1993; 175: 4641-4651Crossref PubMed Google Scholar, 34Mendonca V.M. Klepin H.D. Matson S.W. J. Bacteriol. 1995; 177: 1326-1335Crossref PubMed Google Scholar, 35Schellhorn H.E. Low K.B. J. Bacteriol. 1991; 173: 6192-6198Crossref PubMed Google Scholar, 36Arthur H.M. Lloyd R.G. Mol. & Gen. Genet. 1980; 180: 185-191Crossref PubMed Scopus (82) Google Scholar, 37Feinstein S.I. Low K.B. Genetics. 1986; 113: 13-33Crossref PubMed Google Scholar, 38Morel P. Hejna J.A. Ehrlich S.D. Cassuto E. Nucleic Acids Res. 1993; 21: 3205-3209Crossref PubMed Scopus (68) Google Scholar, 39Washburn B.K. Kushner S.R. J. Bacteriol. 1991; 173: 2569-2575Crossref PubMed Google Scholar, 40Taucher-Scholz G. Hoffman-Berling H. Eur. J. Biochem. 1983; 137: 573-580Crossref PubMed Scopus (26) Google Scholar). The purified enzyme unwinds DNA with 3′ to 5′ polarity (41Matson S.W. J. Biol. Chem. 1986; 261: 10169-10175Abstract Full Text PDF PubMed Google Scholar) and is capable of initiating unwinding from a nick, its presumed biological substrate in the repair pathways (42Runyon G.T. Lohman T.M. J. Biol. Chem. 1989; 264: 17502-17512Abstract Full Text PDF PubMed Google Scholar, 43Runyon G.T. Bear D.G. Lohman T.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6383-6387Crossref PubMed Scopus (94) Google Scholar, 44Brosh Jr., R.M. Matson S.W. J. Biol. Chem. 1997; 272: 572-579Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). UvrD belongs to helicase superfamily I along with otherE. coli helicases such as Rep, RecB, RecD, TraI, and helicase IV (helD gene product) (13Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. FEBS Lett. 1988; 235: 16-24Crossref PubMed Scopus (211) Google Scholar, 16Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1032) Google Scholar), as well as a large number of eukaryotic viral helicases and several yeast helicases. UvrD has previously been the subject of biochemical and genetic studies involving mutation of highly conserved residues in motifs I, II, and III (18George J.W. Robert M. Brosh J. Matson S.W. J. Mol. Biol. 1994; 235: 424-435Crossref PubMed Scopus (81) Google Scholar, 19Brosh Jr., R.M. Matson S.W. J. Bacteriol. 1995; 177: 5612-5621Crossref PubMed Google Scholar, 27Brosh Jr., R.M. Matson S.W. J. Biol. Chem. 1996; 271: 25360-25368Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 44Brosh Jr., R.M. Matson S.W. J. Biol. Chem. 1997; 272: 572-579Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Currently, very little biochemical information exists on the specific role of motif IV in superfamily I and II helicases although its importance for biological function has been demonstrated in genetic studies (45Zhu L. Weller S.K. J. Virol. 1992; 66: 469-479Crossref PubMed Google Scholar, 46Martinez R. Shao L. Weller S.K. J. Virol. 1992; 66: 6735-6746Crossref PubMed Google Scholar, 47Ma L. Westbroek A. Jochemsen A.G. Weeda G. Bosch A. Bootsma D. Hoeijmakers J.H.J. van der Eb A.J. Mol. Cell. Biol. 1994; 14: 4126-4134Crossref PubMed Scopus (45) Google Scholar). In this report, the functional significance of motif IV in E. coli DNA helicase II was addressed by site-directed mutagenesis of arginine residue 284, which is invariant among superfamily I helicases. The results indicated that the mutant protein had a significant decrease in affinity for ATP, implicating motif IV in nucleotide binding. This represents the first helicase mutation outside of motifs I and II that exhibits a severe defect in nucleotide binding. E. coli BL21(DE3) (F− ompT [lon] hsdS BrB− mB− gal dcmλDE3) was from Novagen, Inc.E. coli JH137 (K91lacZ dinD1::MudI (Apr lac)) was obtained from Dr. P. Model (Rockefeller University). BL21(DE3)ΔuvrD and JH137ΔuvrD were constructed previously in this laboratory (18George J.W. Robert M. Brosh J. Matson S.W. J. Mol. Biol. 1994; 235: 424-435Crossref PubMed Scopus (81) Google Scholar). pET81F1+ was from Dr. P. J. Laipis (University of Florida), and pET9d, pET11d, and pLysS were from Novagen, Inc. M13mp7 ssDNA was prepared as described previously (48Lechner R.L. Richardson C.C. J. Biol. Chem. 1983; 258: 11185-11196Abstract Full Text PDF PubMed Google Scholar). Unlabeled nucleotides were from U. S. Biochemicals Corp. Radioactively labeled nucleotides were from Amersham Corp. pET9d-UvrD and pET11d-UvrD were constructed previously in this laboratory (18George J.W. Robert M. Brosh J. Matson S.W. J. Mol. Biol. 1994; 235: 424-435Crossref PubMed Scopus (81) Google Scholar). Restriction endonucleases, DNA polymerase I (large fragment), phage T4 DNA ligase, phage T7 DNA polymerase, and phage T7 polynucleotide kinase were from New England Biolabs Inc. and used as recommended by the supplier. To overexpress helicase II prior to purification, a 10-liter culture of mid-log phase BL21(DE3)/pLysS cells containing pET11d-UvrD or a 2-liter culture of mid-log phase BL21(DE3)ΔuvrD/pLysS cells containing pET9d-UvrD-R284A were induced for protein expression by adding isopropyl β-d-thiogalactopyranoside to 0.5 mm. Growth was continued for 4 h at 37 °C. Wild-type and mutant helicase II proteins were purified using a procedure described previously (49Runyon G.T. Wong I. Lohman T.M. Biochemistry. 1993; 32: 602-612Crossref PubMed Scopus (83) Google Scholar). The concentration of purified protein was determined by spectrophotometric absorbance readings at 280 nm using the previously published helicase II extinction coefficient of 1.29 ml mg−1 cm−1 (49Runyon G.T. Wong I. Lohman T.M. Biochemistry. 1993; 32: 602-612Crossref PubMed Scopus (83) Google Scholar). Storage buffer for helicase II was 20 mm Tris-HCl (pH 8.3), 200 mmNaCl, 25 mm 2-mercaptoethanol, 1 mm EDTA, 0.5 mm EGTA, and 50% glycerol. The plasmid pET81F1-UvrD (27Brosh Jr., R.M. Matson S.W. J. Biol. Chem. 1996; 271: 25360-25368Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), containing the full-length helicase II coding sequence cloned behind a T7 promoter, as well as the phage F1 origin of replication, was the template for site-directed mutagenesis by standard procedures (50Kunkel T.A. Bebenek K. McClary J. Methods Enzymol. 1991; 204: 125-139Crossref PubMed Scopus (633) Google Scholar). The oligonucleotide 5′-GCTGGTAGAGGCGTAGTTTTGCTCC-3′ altered codon 284 of helicase II from CGC(arginine) to GCC(alanine) and disrupted aBsrBI restriction site, allowing the initial screening of mutants by restriction digest. After mutagenesis, a 2.0-kilobase pairNcoI-BsiWI DNA fragment containing the desired mutation was sub-cloned into pET9d-UvrD, replacing the wild-typeNcoI-BsiWI fragment in this plasmid and generating pET9d-UvrD-R284A. The presence of a single mutation at the correct location was verified by sequence analysis of the entire helicase II gene in pET9d-UvrD-R284A using an Applied Biosystems 373A DNA Sequencer. UV irradiation survival was determined as described (19Brosh Jr., R.M. Matson S.W. J. Bacteriol. 1995; 177: 5612-5621Crossref PubMed Google Scholar). The frequency of spontaneous mutant formation was determined as follows. Eleven independent transformants of each of the appropriate cell strains were grown overnight under antibiotic selection at 37 °C. Serial dilutions of each of the saturated cultures were made in M9 minimal media salts. Appropriate dilutions were plated on LB agar to determine cell titer and on LB agar plus 100 μg/ml rifampicin to determine the number of spontaneously arising rifampicin-resistant cells. After incubation at 37 °C for at least 24 h, colonies were counted, and the spontaneous mutant frequency was calculated for each strain by dividing the median value of rifampicin-resistant cells by the average total viable cells. Limited chymotrypsin cleavage of UvrD and UvrD-R284A was performed as described previously (51Chao K. Lohman T.M. J. Biol. Chem. 1990; 265: 1067-1076Abstract Full Text PDF PubMed Google Scholar). α-Chymotrypsin (5 ng) was added to reactions (15 μl) containing either 2 μm UvrD (monomer) or 1.8 μmUvrD-R284A (monomer), and the reaction was incubated for 2 min at room temperature (25 °C). Where indicated, 2 μm M13mp7 ssDNA was included. Reactions were stopped with 15 μl of gel loading buffer (250 mm Tris-HCl (pH 6.8), 3.4% SDS, 1.1m 2-mercaptoethanol, 20% glycerol, 0.01% bromphenol blue) and boiled for 2 min. Products were resolved on a 12% polyacrylamide gel (32:1 cross-linking ratio) in the presence of 0.1% SDS and visualized by staining with Coomassie Brilliant Blue R-250 (Sigma). The binding of UvrD and UvrD-R284A to ssDNA was determined by measuring the retention of [32P]DNA on nitrocellulose filters as described previously (52Matson S.W. Richardson C.C. J. Biol. Chem. 1985; 260: 2281-2287Abstract Full Text PDF PubMed Google Scholar). Reactions (20 μl) contained 25 mmTris-HCl (pH 7.5), 3 mm MgCl2, 20 mm NaCl, 5 mm 2-mercaptoethanol, 50 μg/ml bovine serum albumin, and a [32P]-labeled 92-bp partial duplex helicase substrate (approximately 1.3 μmnucleotide phosphate) (1.86 × 108 cpm μmol−1). Binding reactions were initiated with UvrD or UvrD-R284A over a concentration range of 0.7–84 nm(monomer). After incubation at 37 °C for 10 min, 1 ml of reaction buffer (pre-warmed at 37 °C) was mixed with each sample, and the entire volume was passed through a nitrocellulose filter at a flow rate of 4 ml/min. Filters were washed twice with 1 ml of pre-warmed reaction buffer, dried, and subjected to liquid scintillation counting. Background binding in the absence of enzyme represented less than 0.5% of the total signal and was subtracted from the experimental data. Apparent K d values were calculated as described previously (27Brosh Jr., R.M. Matson S.W. J. Biol. Chem. 1996; 271: 25360-25368Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 53Yong Y. Romano L.J. J. Biol. Chem. 1995; 270: 24509-24517Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Binding of [3H]ATP to UvrD was also examined by nitrocellulose filter binding. Reactions (20 μl) contained 25 mm Tris-HCl (pH 7.5), 3 mm MgCl2, 50 mm NaCl, 6.3 mm 2-mercaptoethanol, 100 μg/ml bovine serum albumin, 12.5% glycerol, and 2.3 μmUvrD or UvrD-R284A (monomer). Binding was initiated by addition of [3H]ATP (1.1 Ci/mmol) to a final concentration of 200 μm at 0 °C. After 4 min, 15 μl of each reaction was applied directly to a nitrocellulose filter presoaked in reaction buffer at 4 °C. Filters were rinsed once with 750 μl of reaction buffer at a flow rate of 4 ml/min, dried, and subjected to liquid scintillation counting. Background binding in the absence of enzyme represented less than 1% of the total signal and was subtracted from the experimental data. The nitrocellulose filters used in the DNA and ATP binding experiments (0.45 μm type HA, Millipore Corp.) were pre-treated by soaking in 0.4 m KOH for 40 min followed by extensive washing with deionized, distilled water. The presence of a dimeric helicase II species was monitored by glutaraldehyde cross-linking as described previously (27Brosh Jr., R.M. Matson S.W. J. Biol. Chem. 1996; 271: 25360-25368Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 49Runyon G.T. Wong I. Lohman T.M. Biochemistry. 1993; 32: 602-612Crossref PubMed Scopus (83) Google Scholar). Reactions (20 μl) contained 20 mm Tricine (pH 8.3), 50 mm NaCl, 5 mm MgCl2, 5 mm 2-mercaptoethanol, 18% glycerol, and either 1.5 μm UvrD (monomer) or 1.4 μm UvrD-R284A (monomer). When present, the oligonucleotide (dT)10 and ATPγS were included at a concentration of 1.7 μm and 3 mm, respectively. Cross-linking was initiated by the addition of glutaraldehyde (EM grade, Electron Microscopy Sciences) to a final concentration of 0.01%. Reactions were incubated at room temperature for 30 min and then quenched with 2 μl of 100 mm lysine acetate and 20 μl of gel-loading buffer (see above). Samples were boiled for 2 min, and the products were resolved on a 9.6% polyacrylamide gel (32:1 cross-linking ratio) in the presence of 0.1% SDS. The DNA unwinding activity of UvrD and UvrD-R284A was determined with 92- and 20-bp partial duplex [32P]DNA substrates prepared as described previously (19Brosh Jr., R.M. Matson S.W. J. Bacteriol. 1995; 177: 5612-5621Crossref PubMed Google Scholar,41Matson S.W. J. Biol. Chem. 1986; 261: 10169-10175Abstract Full Text PDF PubMed Google Scholar). Both substrates were purified by gel filtration on a Bio-Gel A5M column (Bio-Rad) prior to use. Helicase reactions (20 μl) contained 25 mm Tris-HCl (pH 7.5), 3 mmMgCl2, 20 mm NaCl, 5 mm2-mercaptoethanol, 3 mm ATP, and the indicated partial duplex DNA substrate (approximately 1.3 μm nucleotide phosphate) (1.8 × 108 cpm μmol−1 for the 92-bp substrate and 1.6 × 108 cpm μmol−1 for the 20-bp substrate). Reactions were pre-warmed at 37 °C and initiated with the indicated amount of UvrD or UvrD-R284A. After incubation at 37 °C for 10 min, the reactions were quenched with 10 μl of stop solution (37.5% glycerol, 50 mm EDTA, 0.05% each of xylene cyanol and bromphenol blue, and 0.3% SDS). Products were resolved on an 8% non-denaturing polyacrylamide gel, and the results were quantified using phosphor storage technology and ImageQuant software (Molecular Dynamics). The DNA-stimulated hydrolysis of ATP by UvrD and UvrD-R284A was measured as described previously (54Matson S.W. Richardson C.C. J. Biol. Chem. 1983; 258: 14009-14016Abstract Full Text PDF PubMed Google Scholar). ATPase reaction mixtures were identical to those for the helicase reactions with the following exceptions. M13mp7 ssDNA (30 μmnucleotide phosphate) was substituted for the partial duplex helicase substrate, and [3H]ATP was substituted for unlabeled ATP. Reactions (20 μl) were pre-warmed at 37 °C and initiated with the indicated amount of [3H]ATP. Reactions were incubated at 37 °C, and duplicate samples (5 μl) were removed and quenched with 5 μl of stop solution (33 mm EDTA, 7 mm ATP, and 7 mm ADP) after 5 min for UvrD or 10 min for UvrD-R284A. Products were processed as described previously (54Matson S.W. Richardson C.C. J. Biol. Chem. 1983; 258: 14009-14016Abstract Full Text PDF PubMed Google Scholar). DNA helicase II (also called UvrD) has been included, along with a large number of other helicases and putative helicases, in a group designated helicase superfamily I based on conservation of amino acid sequence in seven distinct motifs (13Gorbalenya A.E. Koonin E.V. Donchenko A.P. Blinov V.M. FEBS Lett. 1988; 235: 16-24Crossref PubMed Scopus (211) Google Scholar, 15Hodgman T.C. Nature. 1988; 333: 22-23Crossref PubMed Scopus (323) Google Scholar). Presumably, these motifs represent sites of functional significance that have been evolutionarily conserved. The amino acid sequences of motif IV from theE. coli superfamily I helicases, including UvrD, are shown in Fig. 1. To evaluate the functional significance of motif IV, a mutant uvrD allele was constructed containing an arginine to alanine substitution at position 284 (uvrD-R284A). This arginine is the most highly conserved residue in motif IV. It is found in all identified members of superfamily I. The ability of the mutant protein to substitute for the wild-type protein in two DNA repair pathways was examined in genetic complementation studies. In addition, the UvrD-R284A protein was purified and biochemically characterized. DNA helicase II is an essential component of two DNA repair pathways, methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair (29Husain I. Van-Houten B. Thomas D.C. Abdel-Monem M. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6774-6778Crossref PubMed Scopus (146) Google Scholar, 30Caron P.R. Kushner S.R. Grossman L. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4925-4929Crossref PubMed Scopus (138) Google Scholar, 31Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (446) Google Scholar). The ability of UvrD-R284A to function in each pathway was tested using genetic complementation assays. The frequency of spontaneous mutant formation in E. coli strain JH137ΔuvrD was 161-fold higher than its parent strain JH137 due to loss of a functional methyl-directed mismatch repair system (55Nevers P. Spatz H. Mol. & Gen. Genet. 1975; 139: 133-143Crossref PubMed Scopus (98) Google Scholar, 56Glickman B.W. Radman M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1063-1067Crossref PubMed Scopus (272) Google Scholar). JH137ΔuvrD was also highly sensitive to UV light due to loss of UvrABC-mediated nucleotide excision repair (57Kuemmerle N.B. Masker W.E. J. Bacteriol. 1980; 142: 535-546Crossref PubMed Google Scholar). Plasmids pET9d-UvrD and pET9d-UvrD-R284A were transformed into JH137ΔuvrD to examine the ability of UvrD and UvrD-R284A to restore the wild-type spontaneous mutant frequency and UV resistance. The level of uninduced expression of theuvrD gene from pET9d-UvrD in JH137ΔuvrD was determined previously and was only slightly less than expression from the JH137 chromosome (18George J.W. Robert M. Brosh J. Matson S.W. J. Mol. Biol. 1994; 235: 424-435Crossref PubMed Scopus (81) Google Scholar). This was confirmed in this study (data not shown). The frequency of formation of spontaneous mutants at therpoB locus (Rifr phenotype) was determined as described under “Experimental Procedures.” Wild-typeuvrD, when introduced on the pET9d plasmid, fully restored methyl-directed mismatch repair function as indicated by a relative mutability of 0.6 compared with 1.0 for the parental strain JH137 (Table I). In contrast, the presence of theuvrD-R284A allele in JH137ΔuvrD resulted in a relative mutability of 121, suggesting that the mutant protein did not function in the methyl-directed mismatch repair pathway.Table ISpontaneous mutant frequencies of JH137 and JH137 derivativesStrainRelevant genotypeSpontaneous mutant frequencyRelative mutability× 10 −9JH137uvrD +3.01JH137ΔuvrDΔuvrD485161JH137ΔuvrD/pET9d-UvrDuvrD +1.80.6JH137ΔuvrD/pET9d-UvrD-R284AuvrD-R284A362121The spontaneous mutant frequency was determined as described under “Experimental Procedures.” Relative mutability was obtained by dividing the mutant frequency of each strain by the mutant frequency of JH137. Open table in a new tab The spontaneous mutant frequency was determined as described under “Experimental Procedures.” Relative mutability was obtained by dividing the mutant frequency of each strain by the mutant frequency of JH137. The ability of UvrD-R284A to function in UvrABC-mediated nucleotide excision repair was assessed by exposing cells to UV light and determining survival at increasing UV fluences. Strains JH137, JH137ΔuvrD, JH137ΔuvrD/pET9d, JH137ΔuvrD/pET9d-UvrD, and JH137ΔuvrD/pET9d-UvrD-R284A were compared as shown in Fig.2. pET9d-UvrD completely restored the wild-type level of UV resistance. The mutant allele did not restore wild-type UV resistance and appeared to make the cells slightly hypersensitive to UV light as compared with JH137ΔuvrD. The significance of the latter observation was not explored. It was clear that UvrD-R284A was not able to substitute for UvrD in UvrABC-mediated nucleotide excision repair. To identify the biochemical defects responsible for the loss of biological function of UvrD-R284A, mutant and wild-type helicase II were purified as described previously (49Runyon G.T. Wong I. Lohman T.M. Biochemistry. 1993; 32: 602-612Crossref PubMed Scopus (83) Google Scholar). UvrD was purified to apparent homogeneity as evidenced by the presence of a single protein species on an SDS-polyacrylamide gel (Fig. 3, lane A). Purified UvrD-R284A was contaminated by three faint species migrating slightly faster than UvrD-R284A (Fig. 3, lane D). These species appeared to

Referência(s)