Proc. Natl. Acad. Sci. USA Vol. 82, pp. 4925-4929, August 1985 Biochemistry Involvement of helicase II (uvrD gene product) and DNA polymerase I in excision mediated by the uvrABC protein complex (DNA repair/pyrimidine dimer/"repairosome") PAUL R. CARON*, SIDNEY R. KUSHNERt, AND LAWRENCE GROSSMAN* *Department of Biochemistry, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205; and tDepartment of Molecular and Population Genetics, The University of Georgia, Athens, GA 30602 Communicated by Daniel Nathans, April 5, 1985 ABSTRACT The bimodal-incision nature ofthe reaction of these findings is that the mixture for the in vitro reaction using UV-irradiated DNA catalyzed by the Escherichia coli uvrABC purified uvrA, uvrB, and uvrC proteins lacks important protein complex potentially leads to excision of a 12- to components limiting the repair process to uncoupled repair 13-nucleotide-long damaged fragment. However, the oligonu- reactions. The uvrABC-incised DNA complex is, therefore, cleotide fragment containing the UV-induced pyrimidine dimer amenable to an examination of those other proteins that may is not released under nondenaturing in vitro reaction condi- facilitate the excision reaction. tions. Also, the uvrABC proteins are stably bound to the incised Although the uvrA, uvrB, and uvrC genes are required for DNA and do not turn over after the incision event. In this incision of damaged DNA in vivo, additional gene products communication it is shown that release of the damaged frag- have been implicated in the excision-repair process. Those ment from the parental uvrABC-incised DNA is dependent that have been implicated include the products of the uvrD upon either chelating conditions or the simultaneous addition (12, 13), polA (14, 15), poiC (15), recA (12, 14), recB (12, 14), ofthe uvrD gene product (helicase II) and thepoUl gene product lexA (16), and lig (17) genes. The last four genes control steps (DNA polymerase I) when polymerization of deoxynucleoside beyond the excision step or are the regulatory genes influ- triphosphate substrates is concomitantly catalyzed. The prod- encing the expression of the uvr system. The gene product of uct of this multiprotein-catalyzed series of reactions serves as uvrD has been identified by a number of laboratories as a substrate for polynucleotide ligase, resulting in the restora- helicase II (18-22). DNA polymerase I, the product of the tion of the integrity of the strands of DNA. The addition of the polA gene, participates in excision repair by virtue of its uvrD protein to the incised DNA-uvrABC complex also results coordinated polymerase and 5'- to -3' exonuclease functions in turnover of the uvrC protein. It is suggested that the repair (23) as well as its strong affinity for nicked sites on damaged processes of incision, excision, resynthesis, and ligation are DNA. The involvement of these two proteins in assisting in coordinately catalyzed by a complex of proteins in a "repairo- the release of the damaged fragment and the uvrABC com- some" configuration. plex from irradiated incised DNA is described in this manu- script. The removal of ultraviolet light-induced pyrimidine cyclobu- tane dimers from damaged DNA in bacteria is catalyzed by MATERIALS AND METHODS two essentially different mechanisms. In Micrococcus luteus (1-3) and T4 phage-infected Escherichia coli (4, 5) the Enzymes. The purified uvrA, uvrB, and uvrC proteins were 5'-pyrimidine N-glycosylic linkage ofthe pyrimidine dimer is isolated by methods briefly described previously (10). The initially acted upon by a dimer-specific endonuclease. This helicase II (uvrD) from E. coli was purified according to 17-kDa protein has two activities: A pyrimidine dimer DNA already described methods (19, 24) and the E. coli DNA glycosylase activity initially hydrolyzes the N-glycosyl link- polymerase I, the Klenow fragment of DNA polymerase I, age of the 5'-pyrimidine moiety of the dimer, generating an and bacterial alkaline phosphatase were purchased from apyrimidinic sitejuxtaposed to a thymine-thymidylate dimer. Bethesda Research Laboratories. T4 DNA ligase was ob- A 3'-apyrimidinic acid endonuclease activity associated with tained from Collaborative Research and the E. coli DNA the same enzyme generates a 3'-hydroxylated apyrimidinic ligase was obtained from New England Biolabs. E. coli DNA terminus and a 5'-phosphorylated thymine-thymidylate polymerase III was purified by a modification of a described dimer terminus (3). Although these are seemingly different procedure (25) and was a gift of Jonathan LeBowitz (The activities associated with the same polypeptide, a common Johns Hopkins University). Exonuclease III, T4 DNA poly- modified Michael addition reaction can be invoked to explain merase, and T4 polynucleotide kinase were purchased from both activities (unpublished data). P-L Biochemicals. The incision reaction in uninfected E. coli, which requires Nicking-Reclosing Assay. The uvrABC endonuclease activ- the uvrA, uvrB, and uvrC gene products (6-8), results in two ity was followed by a nitrocellulose filter binding assay after endonucleolytic breaks: one break occurs seven nucleotides denaturation and renaturation essentially as described (10) 5' to a pyrimidine dimer and a second site of hydrolysis is with the exception that the reactions were contained in a three to four nucleotides 3' to the same dimer (9-11). 100-,ul volume and where indicated 1 ,uM each dATP, dCTP, Preliminary evidence suggests a sequential mechanism in dGTP, and dTTP and 5 ,M NAD+ were included in the which the 5' break is made first. Although the size of the reaction mixture. The DNA used in the assay was covalently incised fragment in the DNA suggests that incision may be closed circular DNA (ccc DNA), the double-stranded replica- coordinated with excision reactions, neither fragment release tive form from bacteriophage fd, tritium labeled to a specific under nondenaturing conditions nor turnover of the uvrABC activity of 1.6 x 105 cpm/,ug and containing between 0 and proteins could be demonstrated (9-11). The implication of 12 pyrimidine dimers per DNA molecule as indicated. Each reaction mixture contained 50 ng of fd [3H]DNA and, where The publication costs of this article were defrayed in part by page charge indicated, 880 fmol ofuvrA protein, 800 fmol ofuvrB protein, payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: ccc DNA, covalently closed circular DNA. 4925 Downloaded by guest on September 27, 2021 4926 Biochemistry: Caron et aL Proc. Natl. Acad. Scd USA 82 (1985) 220 fmol of uvrC protein, 200 fmol of uvrD protein, 270 fmol M 1 2 3 4 5 M of DNA polymerase I, and 1400 fmol of T4 DNA ligase. Preparation of 32P-Labeled Substrate DNA. Double-strand- ed fd DNA was digested with BamHI (New England Bio- labs) and labeled by replacement synthesis using T4 DNA polymerase and [a- 2P]dATP (3000 Ci/mmol, Amersham; 1 Ci = 37 GBq) and unlabeled deoxynucleoside triphosphates (26). The resulting DNA (specific activity = 108 cpm/,g) was further purified by precipitation with ethanol and column chromatography through Bio-Gel A-Sm (Bio-Rad). Electrophoretic Identification ofthe Excised Oligonucleotide Fragment. 32P-labeled DNA (10 ng) was allowed to react with the uvrABC protein complex under the standard conditions for 30 min at 370C, followed by precipitation with the addition of 100 Al of 500 mM ammonium acetate/10 mM magnesium acetate/10 ,ug oftRNA per ml and 0.5 ml of isopropyl alcohol at -70°C for 20 min (27). The pellet after centrifugation was resuspended in water and brought to 50 mM ethylenediam- inetetraacetic acid (EDTA). The sample was loaded on a 20% polyacrylamide/7 M urea gel (0.075 x 35 x 50 cm) after addition of an equal volume 98% formamide and heating to 95°C for 5 min. Electrophoresis was carried out for 3 hr at 2000 V. The molecular weight standards used were d(GT- TAAC) (Hpa I linker), (dT)g, and (dT)1o obtained from P-L Biochemicals and were labeled by using polynucleotide kinase and [y-32P]ATP (Amersham). Fragment Release Assay. 32P-labeled DNA was allowed to react with uvrABC according to procedures described in the previous section and loaded directly onto a 0.8-ml DEAE- - Excision pr-odUct Sephacel (Pharmacia) column previously equilibrated with 0.2 M NaCl/50 mM potassium 4-morpholinepropanesulfo- -10 nate (Mops), pH 7.6. The column was washed with 1 ml of0.2 -9 M NaCl/50 mM Mops, pH 7.6, which was found to com- pletely remove mononucleotides from the column. Oligonu- cleotides were eluted with a 3-ml gradient from 0.2 M to 0.5 - 6 M NaCl in 50 mM Mops, pH 7.6, and 300-jul fractions were collected. A (dT)1o standard eluted in fraction 4 and high molecular weight DNA remained bound to the column in 0.5 M NaCl (data not shown). FIG. 1. EDTA-assisted release offragment generated by uvrABC Determination of the Number of Nucleotides Incorporated nuclease on UV-damaged DNA. 32P-labeled DNA was allowed to per Repair Site. The number of nucleotides incorporated per react with uvrABC, precipitated, treated with 50 mM EDTA, loaded repair site was determined by first allowing 10 ng of 3H- onto a 20% polyacrylamide/7 M urea gel, electrophoresed, and labeled fd cccDNA containing an average of one dimer per autoradiographed. Lane 1, (no UV) DNA; lane 2, (no UV) DNA + molecule to react with uvrA, uvrB, and uvrC proteins under uvrAC proteins; lane 3, (no UV) DNA + uvrABC endonuclease; lane the standard reaction conditions at 37°C for 5 min. An aliquot 4, (UV) DNA + uvrAC proteins; lane 5, (UV) DNA + uvrABC was removed and the amount of endonuclease was endonuclease; lanes M, markers of Hpa I linker (6 nucleotides), activity (dT)g, and (dT)1o.