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Enzymatic Properties of Purified Escherichia Coliuvrabc Proteins

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Enzymatic Properties of Purified Escherichia Coliuvrabc Proteins Proc. Nati. Acad. Sci. USA Vol. 80, pp. 6157-6161, October 1983 Biochemistry Enzymatic properties of purified Escherichia coli uvrABC proteins (DNA repair/UV endonuclease/uvrA/uvrB/uvrC) ANTHONY T. YEUNG*, WILLIAM B. MATTES, EUK Y. OH, AND LAWRENCE GROSSMANt Department of Biochemistry, The Johns Hopkdns University School of Hygiene and Public Health, Baltimore, MD 21205 Communicated by Hamilton 0. Smith, June 22, 1983 ABSTRACT The cloned uwrA and uvrB genes of Escherichia METHODS coli K-12 were amplified by linkage to the PL promoter of plasmid uvrC Proteins. pKC30. The uvrC gene was amplified in the high-copy-number Purification of the uvrA, uvrB, and Having plasmid pRLM 24. The three gene products (purified in each case cloned the uvrA, uvrC (12), and uvrB (unpublished data) genes to greater than 95% purity) and ATP are required to effectively from the chromosome of E. coli strain AB1157 we made use of incise UV-damaged DNAs. The uvrABC proteins bind tightly to the strong PL promoter of phage A to amplify the uvrA protein damaged sites in DNA, requiring the initial atachment of the uvrA to 7% of the total protein of E. coli (13) and the uvrB protein protein in the presence of ATP before productive binding of the in a similar manner to 5% of total E. coli protein (unpublished uvrB and uvrC proteins. Using a cloned tandem double insert of data). The uvrC protein was amplified about 300-fold by in- the lac p-o region as a damaged DNA substrate for the uvrABC creasing the uvrC gene dosage through its cloning in high-opy- complex and analyzing the incision both 5' and 3' to each pyrim- number plasmid pRLM24 (source: Roger L. McMacken, The idine dimer, we found that one break occurs 7 nucleotides 5' to Johns Hopkins University). The uvrA protein has been purified a pyrimidine dimer and a second break is made 3-4 nucleotides to >95% purity by using three chromatographic steps: (i) Affi- 3' from the same pair of pyrimidines in the dimer. No such breaks Gel blue (Bio-Rad), (ii) phosphocellulose P-li (Whatman), and are found in the strand complementary to the dimer. The size of (iii) single-stranded DNA-agarose affmity chromatography (Fig. the incised fragment in the DNA suggests that incision may be co- 1). The uvrB protein was purified to >95% purity in four chro- ordinated with excision reactions in repair processes. matographic steps: (i) Affi-Gel blue, (ii) DEAE-Sephacel (Phar- macia), (iii) phenylagarose, and (iv) Sephadex G-150 (Phar- There are at least two mechanistic responses to UV-induced .macia) (Fig. 1). The uvrC protein was purified to >95% purity pyrimidine cyclobutane dimers in DNA, depending on the bi- by three chromatographic steps: (i) Affi-Gel blue, (ii) phospho- ological systems examined. The least complicated mechanism cellulose P-li, and (iii) single-stranded DNA-agarose affinity occurs in those UV-resistant organisms such as Micrococcus lu- chromatography (Fig. 1). teus or T4 phage-infected Eschenchia coli. In these cell systems The uvrABC UV-Endonuclease Assay. The uvrABC endo- the 5'-pyrimidine N-glycosylic linkage of the pyrimidine dimer nuclease assay employed in these experiments exploits the abil- is acted on by a unique dimer DNA glycosylase, generating an ity of nitrocellulose filters to discriminate between replicative apyrimidinic site juxtaposed to a thymine-thymidylate dimer form (RF)-I DNA species and the single-stranded components (1-5). This unusual site is recognized by a 3'-apyrimidinic site of the enzyme-generated RF-II DNA (15). The assay conditions endonuclease specifically generating a hydroxylated 3'-apyrim- were essentially those described by Seeberg (8, 9), and in- idinic site and a 5'-phosphorylated thymine-thymidylate dimer cluded in a 140-4ul reaction volume are 85 mM KCI, 40 mM site (1). The dimer-specific endonucleases from both M. luteus potassium morpholinopropanesulionate (Mops) at pH 7.6, 1 mM and T4-infected E. cob are bifunctional proteins possessing both NaEDTA, 1 mM dithiothreitol, 15 mM MgSO4, 2 mM ATP, 39 the DNA glycosylase and the 3'-apyrimidinic site endonuclease fmol (0.16 ug) of 3H-labeled phage fd DNA (5 x 104 cpm/,ug), activities (1, 6, 7). 490 fmol of uvrA protein, 535 fmol of uvrB protein, and 516 The uvr-encoded system necessary for incision in E. coli is fmol of uvrC protein. The mixture is incubated at 37TC. The composed of the uvrA, uvrB, and uvrC gene products and in- number of pyrimidine dimers in each fd DNA molecule av- cises not only UV-irradiated DNA but also DNAs affected by eraged between 1 and 6 according to the needs of different ex- a variety of agents that form "bulky" adducts. Incision by the periments. uvrA, uvrB, and uvrC gene products in vitro was demonstrated Assembly of the uvrABC Complex. The mechanism of by Seeberg (8, 9) through the complementation of various uvr- uvrABC protein-DNA complex formation will be the subject of mutant extracts with partially purified uvr proteins. Further a forthcoming publication. The amount of fd DNA that has elaboration of these findings was achieved through the cloning, uvrABC proteins associated as a protein-DNA complex was expression, and amplification of the three uvr genes, and the measured by the nitrocellulose filter binding assay (15). The site of their catalytic action was subsequently assessed on UV- assembly reaction conditions were identical to those employed irradiated DNA fragments of defined sequence (10, 11). It was for studying incision assays. from these studies that Rupp and his colleagues suggested that Preparation of Labeled DNA Fragments. All uvrABC in- the uvrABC endonuclease complex generates two breaks on cision reactions monitored by DNA sequence analysis em- either side of a dimer site. It is the purpose of this commu- ployed four separate substrates derived from the double lac nication to show that the highly purified uvrA, uvrB, and uvrC Abbreviations: bp, base pair(s); DNG, dimer N-glycosylase; RF, rep- gene products act bifunctionally and that the active complex licative form. "Dimer" indicates a cyclobutane dimer of T-T, C-C, C-T, also hydrolyzes single phosphodiester bonds. or T-C combinations; TOT, COC, COT, and TOC are the symbols for the same photoproducts. The publication costs of this article were defrayed in part by page charge * Present address: Institute for Cancer Research, Fox Chase Cancer payment. This article must therefore be hereby marked "advertise- Center, Philadelphia, PA 19111. ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 6157 Downloaded by guest on October 2, 2021 6158 Biochemistry: Yeung et al. Proc. Natl. Acad. Sci. USA 80 (1983) A 1 5 4 32 B 6 54 3 2 1 C 5 4 3 2 1 uwA-o _ Q V - 94 -a i _94 ........... uvr8 94 67 am i67 6 uvrC- -- 43 -0 43 3043 30 _ Z 30 20 _ _ 20 20_2 FIG. 1. Purification of the uvrA, uvrB, and uvrC proteins. Purification procedures for the uvrA, uvrB, and uvrC proteins were monitored by sodium dodecyl sulfate/9% polyacrylamide gel electrophoresis according to the method ofLaemmli (14). (A) Purification ofthe uvrA protein. Lane 1, molecular weight standards, labeled x 10-3; lane 2, crude extract of 420C-induced GHY8533 in which uvrA was expressed from the clonedPL promoterofA; lanes 3,4, and 5, pooled uvrA protein peakfractions fromthe Affi-Gel bluecolumn, phosphocellulose P-11 column, and single-stranded DNA-agarose columns, respectively. (B) Purification of the uvrB protein. Lane 1, molecular weight standards; lane 2, 420C-induced BM1731 cells in which the uvrB gene is expressed from the clonedPL promoter of A; lanes 3, 4, 5, and 6, pooled uvrB protein peak fractions from the Affi-Gel blue column, DEAE-Sephacel column, phenyl-agarose column, and Sephadex G-150 column, respectively. (C) Purification of the uvrC protein. Lane 1, molecular weight standards; lane 2, crude extract of EOH-013 in which uvrC was amplified by its cloning in pRLM 24; lanes 3, 4, and 5, pooled uvrC protein peak fractions from the AfFi-Gel blue column, phosphocellulose P-11 column, and single-stranded DNA-agarose column, respectively. p-o DNA fragment: the 5'-labeled 117-base-pair (blp) fragment, lac p-o was excised from pLJ3 (17) and inserted into the unique the 3'-labeled 117-bp fragment, the 5'-labeled I[68-bp frag- EcoRI site of the replication control-defective plasmid pRLM ment, and the 3'-labeled 168-bp fragment (Fig. 2). Because the 24 to produce pPYC3. The E. coli C-600 strain containing the DNA sequence in the original 285-bp fragment is a tamdem head- plasmid was grown in Luria broth to stationary phase in the to-tail repeat, the use of these four labeled fragnnents allows- presence of kanamycin at 20 ,g/ml and the plasmids were iso- analyses of breaks on the strand complementary to the position lated as described by Tanaka and Weisblum (18). To avoid the of a UV-induced pyrimidine dimer as well as breaks c)n the strand use of UV light required for visualizing fragments during the containing the dimer. isolation, 125 ,ug of EcoRI-digested plasmid was 5'-end labeled Defined DNA fragments were prepared and labeled ac- by treatment with bacterial alkaline phosphatase (Bethesda Re- cording to procedures described by D'Andrea anid Haseltine search Laboratories) for 2 hr at 60°C followed by polynucleotide (16) with modification. To prepare large amounts of this DNA kinase and [y-32P]ATP (19). The labeled reaction mixture was fragment for study, the 285-bp EcoRI fragment containing the mixed with 1 mg of EcoRI-hydrolyzed pPYC3 and then sep- arated on a 6% nondenaturing polyacrylamide gel.
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