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Effect of DNA Polymerase I and DNA Helicase II on the Turnover Rate Of Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6774-6778, October 1985 Biochemistry Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease (UvrA, -B, -C, -D proteins/pyrimidine dimers/cisplatin) INTISAR HUSAIN*, BENNETT VAN HOUTEN*, DAVID C. THOMAS*, MAHMOUD ABDEL-MONEMt, AND AZIZ SANCAR* *University of North Carolina, School of Medicine, Department of Biochemistry, Chapel Hill, NC 27514; and tMax-Planck-Institut fur Medizinische Forschung, Abteilung Molekulare Biologie, Heidelberg, Federal Republic of Germany Communicated by Mary Ellen Jones, June 20, 1985 ABSTRACT UvrABC excision nuclease (UvrA, UvrB, and is in reasonable agreement with the 0.12-0.50 min-1 that can UvrC proteins) of Escherichia coli removes nucleotide mono- be calculated from the in vivo data published by several and diadducts from DNA in the form of oligonucleotides 12 or workers (5, 13-15). 13 bases long. We find that the purified enzyme dissociates from DNA very slowly, if at all, in the absence ofother proteins MATERIALS AND METHODS implicated in excision repair. Addition of DNA polymerase I and helicase II (UvrD protein) to the reaction mixture stimu- Enzymes. The UvrA, UvrB, and UvrC subunits of lates the turnover rate of the excision nuclease to a level UvrABC excision nuclease were purified separately as de- comparable to that observed in vivo. scribed previously (1, 16). The UvrD protein (DNA helicase II) was purified according to the original protocol of Abdel- The Escherichia coli UvrABC excision nuclease is an ATP- Monem et al. (17) using as a starting material a strain carrying dependent DNA repair enzyme that removes oligomers the uvrD gene on a multicopy plasmid (18). The detail of this containing modified nucleotides. The enzyme is made up of purification procedure will be published elsewhere. The pol three subunits, UvrA (Mr, 103,749), UvrB (Mr, 84,000), and I (nuclease free) and the Klenow fragment were obtained UvrC (Mr, 66,038). The three proteins acting in concert from Boehringer Mannheim, and E. coli DNA ligase and T4 hydrolyze the eighth phosphodiester bond 5' and the fourth DNA polymerase from New England Biolabs. The E. coli or fifth phosphodiester bond 3' to the damaged nucleotide(s) Rep protein was kindly supplied to us by J. Hurwitz and G. (1). It has been reported that uninduced wild-type E. coli cells Goetz; T4 DNA ligase was a gift from J. Griffith. Polyclonal contain about 20 molecules of UvrA (2), 140 molecules of antibodies against DNA helicase II were prepared by stan- UvrB (3), and 10 molecules of UvrC (4). While these dard methods; they were nuclease free and did not cross react estimates were based on indirect measurements, it is unlikely with any of the proteins used in our reconstruction studies. that there would be more than 100 enzyme complexes per All of the proteins used in our work were greater than 95% cell, which poses an interesting dilemma with regard to pure as judged by analysis on Coomassie blue-stained excision repair. It is well established that following UV NaDodSO4/polyacrylamide gels. Protein concentrations irradiation E. coli cells remove several thousand pyrimidine were determined by the Bradford assay. dimers when held in buffer (see ref. 5), yet recent experi- Substrates. Radiolabeled or unlabeled pBR322 DNA was ments with purified enzyme suggest that the enzyme may not prepared by standard methods and superhelical DNA was turn over-i.e., dissociate from the DNA (6). It is, therefore, purified through two successive ethidium bromide/CsCl den- expected that additional factors stimulate the turnover rate of sity gradient centrifugations (19). The tritiated DNA used in the the enzyme in vivo. Two likely candidates are E. coli DNA incision and filter binding assays had a specific activity of 1.5 x polymerase I (pol I) and DNA helicase II (UvrD protein) 105 cpm/,ug. The substrate for these assays was prepared by because extensive in vivo data indicate that these two irradiating the DNA with 254-nm UV light that produced 10 proteins are involved in excision repair (7-10). Moreover, it lethal hits per molecule as measured by the transformation has been found that UvrD protein stimulates UvrABC assay (19). The DNA used for the incision and filter binding excision nuclease in cell-free extracts (11) or partially purified assays contained 70-75% superhelical molecules. The substrate excision nuclease preparations (12). However, it was not for the excision assay was prepared as described (20). clear these studies whether UvrD stimulated Assays. The activity and turnover rate ofUvrABC excision from protein the nuclease were measured by three separate assays: incision, rate of excision nuclease-DNA complex formation or the excision, and filter binding. All the assays were conducted in turnover rate of the enzyme. a nucleotide-excision-repawr buffer which contained 50 mM In this communication using proteins purified to near Tris-HCI, pH 7.4/50 mM KCl/10 mM MgCl2/2 mM ATP/33 homogeneity we demonstrate that helicase II alone has no ,uM each of dATP, dGTP, dTTP, and dCTP/10 mM dithio- effect on the initial rate ofexcision nuclease activity and only threitol/10% glycerol (vol/vol)/bovine serum albumin at 50 a small effect on its turnover. Similarly we find that pol I ,g/ml plus DNA and repair proteins at the amounts indicated. alone does not stimulate the ABC excision nuclease. How- The incision assay measures the conversion of superhelical ever, the combination of the two causes the enzyme to turn DNA to open circles and has been described elsewhere (19). over such that in a complete excision repair system (UvrABC The excision assay measures the removal of radioactive excision nuclease, helicase II, pol I, DNA ligase) the rate of adducts [in this case cis-1,2-diamino[4,5-3H]cyclohexanedi- the removal of nucleotide adducts approaches 0.08 adduct chloroplatinum(II) ([3H]cisplatin) adduct] from an otherwise per UvrABC excision nuclease complex per min. This value unlabeled DNA and has been described in detail (20). The filter binding assay is the classical nitrocellulose filter binding The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: cisplatin, cis-1,2-diamino[4,5-3H]cyclohexanedichlo- in accordance with 18 U.S.C. §1734 solely to indicate this fact. roplatinum(II); pol I, E. coli DNA polymerase I. 6774 Downloaded by guest on October 1, 2021 Biochemistry: Husain et al. Proc. Natl. Acad. Sci. USA 82 (1985) 6775 assay adapted to the UvrABC excision nuclease complex .- -1 -- ~~~~~----- - 14 A (21). Since both UvrA and UvrC proteins bind to DNA in the -i- S ... .*- A - C, _ i4.- r ~ ~ 0c absence ofcomplex formation, there is a background that can I,_ ' *. 02- . be eliminated by removing uncomplexed UvrA and UvrC -4 -- wZ"0 ..% I IC. proteins with short incubation (5 min) with an excess of MI unlabeled UV-irradiated DNA. We have established that UvrABC excision nuclease-DNA complexes have consider- ably longer half-lives than either UvrA-DNA or UvrC-DNA complexes. The UvrA and UvrC molecules that are not in the -7 ,Iw. excision nuclease complex therefore bind the excess c unlabeled DNA before filtering and do not bind the labeled I DNA to the filter. Thus, under these experimental conditions the amount of radioactive DNA retained on the filters is a measure ofthe amount ofexcision nuclease-DNA complexes plus UvrAB-DNA complexes present at the time offiltration. 2-(IC C~r _-x ~~_ _ ---_ U* RESULTS Cf1 )4~~~~~~~. A Defimed in Vitro System for Excision Repair. The current 2 model for nucleotide excision repair in E. coli involves the 4.~~~~0. excision of a 12- or 13-base-long oligonucleotide by UvrABC 0042I excision nuclease and dissociation of the DNA-protein complex, filling in the gap by polI and sealing it by ligase (1). 026 However, both in vivo (8-10) and in vitro (11, 12) data 42 4 indicate that the UvrD gene product (18, 22-24), helicase II (17), is also involved in dimer removal. Therefore, for total reconstitution of excision repair in vitro and definition of the functions and interactions of all components, it is necessary to purify all the potential excision repair proteins and study the effects ofeach individually and in combinations. We have obtained the E. coli excision repair proteins in nearly homo- geneous form and utilized them in our reconstitution exper- iments. In Fig. 1 we present a NaDodSO4/polyacrylamide gel TIMEfE in) of the proteins used in our study. This gel contains all of the "/= E. coli proteins presumed to be necessary for excision repair FIG. 2. Effects of pol I and helicase II on UvrABC excision with the exception of DNA ligase. In our studies we used T4 nuclease as measured by the incision assay. (Left) Reaction kinetics. (Right) Photographs of the agarose gels from which the data for the DNA ligase instead ofE. coli DNA ligase because the former graphs in the left panel was obtained. (A) Incision by UvrABC enzyme was made available to us in large quantities. How- excision nuclease. (B) Incision by UvrABC excision nuclease plus ever, we have carried out some of the critical experiments pol I. (C) Incision by UvrABC excision nuclease plus DNA helicase reported in this study with both enzymes and have obtained II. (D) Incision by UvrABC excision nuclease in the presence of pol essentially the same results; therefore, the conclusions we I plus helicase II. Ligase was present in the reactions B-D. The inset draw from our experiments with T4 ligase should be valid for in D shows the initial part of the reactions of A and D in a more excision repair in E.
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