THE JOURNALOF BIOLOGICAL CHEMISTRY Vol. 262, No. 27, Issue of September 25, pp. 13180-13187,1987 0 1987 by The American Societyfor Biochemistry and Molecular Biology, Inc. Printed in U.S.A. DNase I Footprint of ABC Excinuclease” (Received for publication, April 3, 1987) Bennett Van HoutenSg,Howard Gamperll((**,Aziz SancarS, and JohnE. HearstllJJ$$ From the $Department of Biochemistry, University ofNorth Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27514, the TDepartment of Chemistry, University of California, Berkeley, California 94720, and the ((Divisionof Chemical Biodynamics, Lawrence Berkeley Laboratory, Berkeley, California 94720 The incision and excision steps of nucleotide excision In Escherichia coli, the initial steps of nucleotide excision repair in Escherichia coli are mediated by ABC exci- repair are mediated by the enzyme ABC excision nuclease nuclease, a multisubunit enzyme composed of three (ABC excinuclease) which is composed of threeproteins, proteins, UvrA, UvrB, and UvrC. To determine the UvrA (Mr= 103,874), UvrB (M, = 76,118), and UvrC (M, = DNA contact sites and the binding affinity ofABC 66,038) (Husain et al., 1986; Arikan et al., 1986; Backendorf excinuclease for damaged DNA, it is necessary to en- et al., 1986; Sancar, G. et al., 1984). These subunits function gineer a DNA fragment uniquely modified at one nu- cleotide. We have recently reported the constructionof in a concerted manner to hydrolyze the 8th phosphodiester a 40 base pair (bp) DNA fragment containing a psora- bond 5’ and the 4th or 5th phosphodiester bond 3‘ to a len adduct at a central TpA sequence (Van Houten, B., modified nucleotide(s). ABC excision nuclease has broad sub- Gamper, H., Hearst, J. E., and Sancar, A. (1986a) J. strate specificity acting on UV-induced pyrimidine dimers Biol. Chem. 261, 14135-14141). Using similar meth- and 6-4 photoproducts, cisplatin diadducts, N-acetoxyace- odology a 137-bp fragment containinga psoralen-thy- tylaminofluorene, and psoralen monadducts with the same mine adduct was synthesized, and this substrate was incision motif, regardless of the size or the conformation of used in DNase I-footprinting experiments withthe sub- the modified nucleotides (Sancar and Rupp, 1983; Yeung et units of ABC excinuclease. It was found that theUvrA al., 1983; Sancar et al., 1985; Beck et al., 1985, Van Houten et subunit bindsspecifically to thepsoralen modified 137- al., 1986a). The notable exception to this rule is the mode of bp fragment withan apparentequilibrium constant of action ofABC excision nuclease on psoralen cross-linked K, = 0.7 - 1.5 X lo8 M-’, while protecting a 33-bp DNA in which the dual incisions are made at the 9th and3rd region surrounding the DNA adduct. The equilibrium phosphodiester bonds 5‘ and 3‘ (respectively) to thethymine constant for the nonspecific binding of UvrA was K, which is covalently attached to thefuran-side of the psoralen = 0.7 - 2.9 X lo5M” (bp). In thepresence of the UvrB molecule (Van Houten et al., 1986b). This highly conserved subunit, the binding affinityof UvrA for thedamaged incision pattern, in addition to thebroad substrate specificity substrate increased to K. = 1.2 - 6.7 X lo8 M” while the footprint shrunk to 19 bp. In addition the binding of ABC excinuclease strongly suggests that theenzyme binds of the UvrA and UvrB subunits to the damaged sub- to a helical distortion common to most bulky DNA adducts strate caused the 11th phosphodiester bond 5‘ to the and not to theparticular modified nucleotide (Van Houten et psoralen-modified thymine to become hypersensitive al., 1986b). to DNase I cleavage. These observations provide evi- A model for the action mechanism of ABC excinuclease has dence of an alteration in theDNA conformation which been proposed (Husain et al., 1985; VanHouten et al., 1986a). occurs during the formation of the ternary UvrA. The UvrA subunit is an ATPase which uses the binding and/ UvrB .DNA complex.The additionof the UvrC subunit or hydrolysis of ATP to facilitate specific binding to damage- to the UvrA. UvrB-DNA complex resulted in incisions induced deformities in the DNA helix. The UvrB subunit in on both sides of the adduct but did not cause any association with the UvrA subunit forms a stable ternary detectable changein the footprint. complex with DNA.UvrC interacts with this complex to Experiments with shorter psoralen-modified DNA initiate the dual DNA incisions. Genetic and biochemical data fragments (20-40 bp) indicated that ABC excinuclease suggest that other organisms including yeast, Drosophilin, and is capable of incising a DNA fragment extendingeither man, contain a nucleotide excision repair pathway analogous 3 or 1 bp beyond the normal 5’ or 3’ incision sites, respectively. These results suggest that the DNA be- to that found in E. coli (reviewed by Friedberg, 1985). ABC yond the incision sites, while contributing toABC ex- excinuclease therefore representsa unique system for the cinuclease-DNAcomplex formation, is not essential for investigation of protein-DNA interactions which occur during cleavage to occur. the process of DNA damage detection and repair. An important step in determiningthe mechanism by which * This work wassuppor+ed by National Institutes of Health Grants ABC excinuclease locates, binds to, and incises DNA sur- GM11180 and GM32833 and National Science Foundation Grant rounding an adducted nucleotide(s) is to determine the con- PCM8351212. The costs of publication of this article were defrayed tact sites andbinding affinities of the individual Uvr subunits in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. to a damaged DNA substrate. DNase I-footprintingtech- Section 1734 solely to indicate this fact. niques have been widely exploited in determining these pa- Recipient of National Institutes of Health Postdoctoral Fellow- rameters for several types of protein-DNA interactions (Galas ship GM11277. and Schmidt, 1978; reviewed by Brenowitz, 1986). The main ** Postdoctoral fellow supported by National Institute of General limitation of DNase I-footprinting experiments with DNA Medical Sciences Grant GM11180. $$ Support was received in part from the Department of Energy, repair proteins has been the availability of a DNA fragment Office of Health and Environmental Research Contract DE-ACO3- containing one DNA adduct located at a defined position. We 76F0093. have recently employed oligonucleotide synthesis and psora- 13180 Footprint of ABC Excinuclease 13181 len photochemistry to engineer a 40-bp' DNA fragment with threitol, 2 mM ATP, and 100 pg/ml of bovine serum albumin (DC buffer). a 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) furan- Maximum subunit binding and incision were obtained when the side monoadduct at a central thymine (Van Houten et al., subunits (0.1-30 pmol) were preincubated for 5 min at 37 "C prior to 1986b). This substrate was useful in an unequivocal determi- the addition of substrate DNA (0.1-0.3 pmol). After the subunits nation of the incision mechanism ofABC excinuclease for were allowed to equilibrate with the DNA for 30 min at 37 "C, the psoralen monoadducts and cross-links but proved to be of reaction mixtures were cooled to room temperature and were made insufficient length for DNase I experiments. Using the same 2.5 mM in CaCl,. DNase I (0.5 ng, Bethesda Research Laboratories) was added, and after 5 min at room temperature the reactions were methodology we have constructed a 137-bp DNA fragment stopped by the addition of EDTA to a final concentration of 15 mM. uniquely modified with psoralen and used this DNA as a After rapidly freezing, the mixtures were lyophilized to dryness, 50 pl substrate for ABC excinuclease binding in DNase I-footprint- of formamide-plus-dyes were added, and the samples were heated at ing experiments. 90 "C for 2 min followed by quick cooling on ice. Portions of the We report here that the UvrA protein is the DNA damage samples (2-5 pl) were applied to an 8% polyacrylamide-sequencing gel and electrophoresed for 2.5 h at 1200 V and 25 milliamps. The gel recognition subunit and that the binding of UvrB to the was dried and was exposed to x-ray film (Kodak, GB-X2 film) UvrA.DNA binary complex induces tighter binding and is overnight at -70 "C with an intensifying screen. accompanied by a change in the conformation of the DNA- Determination of the Binding Affinities-The quantitation by op- enzyme complex. The addition of UvrC, whileresulting in the tical density of individual DNase I bands was performed by densito- dual DNA incisions, does not seem to change the binding metric scanning of appropriately exposed autoradiograms. The auto- specificity or nature of the UvrA-UvrB footprint. Additional radiograms were scanned on an Optronics P-1000 film scanner with an AED graphics terminal. Data analysis was performed on a VACS data from experiments with short psoralen-modified DNA 11/730 computer using an algorithim, Gelscan, developed by Frank fragments (20-40 bp) suggest that the DNA flanking the Hage of the Protein Crystallography Facility at the University of DNase I footprint helps to stabilize the formation of the North Carolina. This program is similar to theone recently described active ABC excision nuclease but is not absolutely required by Brenowitz et al. (1986). The percent saturation of UvrA binding for DNA incisions to occur. was determined from the relative intensity of the integrated optical density for the bands of interest. The protein concentration which MATERIALS ANDMETHODS reduced the band intensity to 50% was taken as theKd- Incision Efficiency on Minimal Length Substrates-Terminally la- DNA Oligonucleotides-Oligomers (30-60-mers) used for the con- beled 40-bp substrate or gel-purified restriction fragments of this struction of the 137-bp substrate were a gift from Applied Biosystems substrate containing the psoralen-modified thymine (0.2-0.5 pmol) (Foster City, CA), while the oligomers used for modification by HMT were treated with UvrA (5.0 pmol), UvrB (10 pmol), and UvrC (10 and for the construction of the 40-bp substrate were synthesized by pmol) in ABC buffer for 30 min at 37 'C.