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. The reaction mixtures were phosphotriester chemistry using a Biosearch instrument. Full length frozen, lyophilized, resuspended in formamide-plus-dyes and loaded oligomers were purified on 7 M urea 12% or 20% polyacrylamide- onto 12% polyacrylamide-sequencing gels. The intact and the ABC sequencing gels. excision nuclease-generated bands were located by autoradiography HMT-modified Oligomers-The psoralen-monoadducted octamer and excised from the gel. The amount of DNA in each band was or dodecamer were obtained as described previously (Van Houten et determined by Cerenkov radiation, and theincision efficiency of ABC al., 1986a). Briefly, the 5'-phosphorylated 8-mer, TCGTAGCT (175 excinuclease was determined from the ratio of incised DNA compared pg) and a complementary 5-phosphorylated 12-mer, GAAGCTAC- to thetotal. GAGC (250 pg) were added together in buffer (0.7 ml) containing 100 mM NaCl, 10 mMMgC12, 1.0% ethanol, and 21 pgof HMT (HRI RESULTS Associates, Berkeley, CA). The two oligomers were cross-linked by exposure to 320-380 nm ultraviolet light (600 milliwatts cm-') for 3 Design and Preparation of 137-bp HMT-modified Frag- min at 4 "C. A second aliquot of HMT was added, and the UV ment-The construction of a DNA fragment containing a exposure was repeated. The reaction mixture was extracted with modified nucleotide at a defined position was essential for chloroform/isoamyl alcohol (19:l). followed by an etherextraction to DNase I-footprinting experiments with ABC excinuclease. remove the unreacted HMT,and the DNA was precipitated in Psoralens exhibit amarked preference for photoreaction with ethanol. The two orientational isomers of the HMT cross-linked 8- mer and 12-mer were purified by electrophoresis on a 7 M urea 20% DNA containing a TA sequence, and as reported previously, polyacrylamide gel. To obtain the furan-side-monoadducted octamer we have used this property of psoralens in the preparation of or dodecamer, the cross-linked oligomers were partially photoreversed oligonucleotides modified at a central thyminewith the furan- by exposure to a Sylvania model G30T8 germicidal UV lamp (254 side monoadduct of HMT (Van Houten et al., 1986a). These nm). The monoadducted oligomers were purified by gel electropho- oligonucleotides were used in ligation reactions to construct resis as described above. uniquely modified 40- and 137-bp DNA fragments (Fig. 1). Preparation of the 40- and 137-bp DNA Substrates-The purified psoralen furan-side-monoadducted octamer or dodecamer was ligated Previous DNase I-footprinting experiments with ABC exci- with a series of complementary oligomers to obtain the 40- or 137-bp nuclease and the 40-bp DNA fragment indicated that this DNA fragments (respectively) shown in Fig. 1. The endlabeling and substrate was of insufficient length to determine the contact ligation reactions were as reported previously (Van Houten et al., sites of the Uvr subunits,and we therefore designed and 1986a). Full length DNA duplexes were obtained by reannealing the constructedthe 137-bp fragment. Restriction fragments of appropriate length single strands (138 or 41 bases) which had been the 40-bp substrate were used to determine the minimum purified on denaturing gels. This purification step was absolutely length substratefor ABC excinuclease digestion. required to obtain DNA molecules that were completely double stranded. The identity and integrity of the full length duplexed DNA Psoralen-induced DNA Conformational Changes-DNase I were checked by restriction enzyme digestions and Maxam-Gilbert has been used as a probe for conformational changes in the sequencing (1980). A routine yield of fully duplexed substrate was 3- DNA helix which occur during the binding of certain drugs to 6 pg from a total of 36 pg of component oligomers. The specific DNA (Drew andTravers, 1985). The width of the minor activity of the DNA fragments used in these experiments was 0.5 - groove is believed to be an important determinant in the 5.0 X lo7cpmlpg. selection of a cleavage site by DNase I, with an optimal width DNase I Footprinting of the Uvr Subunits-ABC excinuclease of 12 (Drew and Travers,1984). The binding of echinomycin reactions were routinely performed in 50-rl reactions containing 50 A mM KCI, 50 mM Tris-HC1, pH 7.5, 10 mM MgCl,, 10 mM dithio- and distamycin to DNA are known to alter thewidth of the minor groove and in turn have been shown to affect the The abbreviations used are: bp, base pairs; HMT, 4'-hydroxy- DNase I cleavage pattern for certain sequences (Low et al., methyl-4,5',8-trimethylpsoralen. 1986). Modification of the thymine at position 74by the 13182 Footprint of ABC Excinuclease

A)

I IO 20 30 60 .O 50 70 BO 90 100 ,IO 120 130 I...... l,..l.. 1. .?...... S' - CCCI\TCCCCCTC~GI\CMTTMTCATCCGCTCCTATAATCTCTCCAA~CCACACTCAGT~CG~~CC~GC~ACG~CCTC~CCCC~TCC~~~CGTCT~CATCTCCC~ATAGTC~GTCCT~TTAATTTCC~CC~- 3' 3' - ~G~~~~~~~~~CTGTTAA~~~~~~~~~~GCATA~~~~~~~~~~~~~G~CTC~~~~~~~~~~~~~TTC~~~~~~~~~~~~~~~T~C~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~~~~~~~. 5, A A

81

1 10 20 30 40 . . . .v. . . . 5' - CTATCCATGGCCTCCAGTCGTACCTGM~CCTACTGAGTC - 3' 3' - ATAGCTACCGGACGTCAG~~TCGAC=~CCI\TG*CTCAC~- 5' A A FIG. 1. Nucleotide sequences of the psoralen-monoadducted DNA substrates. The DNA substrates were constructed from several component oligomers. The sitesof ligation are indicated by arrows. The central top strand oligomer was modified with the furan-side monoadduct of HMT at an internal thymine, so that in both substrates the top strand of the full length duplex is always the psoralen-modified strand. The DNA fragments contained multiple restriction enzymes sites and 5' overhanging ends. A, the 137-bp DNA fragment composed of eight oligomers; the HMT-modified thymine is indicated by a filled circle at position 74. E, the 40-bp DNA fragment composed of six oligomers; the HMT-modified thymine is indicated by a filled circle at position 20. furan-side monoadduct of HMT produced a significant change and K, = 0.7 - 2.9 X lo6 M" bp, respectively. Based on these in the DNase I digestion pattern in the vicinity of the DNA equilibrium constants an estimated 4.3 kcal increase in free adduct (compare positions 70-80, lanes 1 and 8 in Fig. 2, A energy is associated with the specific binding of UvrA to the and B). The most striking change was the complete disap- substrate. Kinetic studies have suggested that ABC excinu- pearance of the DNase I cleavage site between C,, and T74 clease consists of one of each subunit (Husain et aZ., 1985), (band II, lanes 1 and 8, Fig. 2A) in the top strand.This while recent evidence from DNA unwinding experiments (Oh particular site was predicted by Pearlman et al., (1985) to be et al., 1986) and hydrodynamic studies2 suggest that theactive unwound by -7.5" as compared to normal B-DNA, and could form of UvrA may be a dimer at the concentrations used in be expected to change the minor groove width making this an these experiments. Since the exact composition of the active unfavorable cleavage site for DNase I. In addition the HMT- form of the ABC excinuclease is not known we have reported modified thymine also caused a reduction in the cleavage of the equilibrium constants of UvrA binding in terms of con- the phosphate bonds at positions G73'-A74' and C77'-T7s' and centration of UvrA as a monomer. an enhancement in DNase I cleavage at T75'-G76' in the The footprinting experiments in Fig. 2 revealed two addi- bottom strand (bands I1 + 111, respectively, of lanes 1 and 8, tional features of Uvr A binding: 1) there was an apparent Fig. 2B). increase in the region protected with increasing Uvr A con- DNase I Footprint Titrations of UvrA-The UvrA subunit centration, 2) the footprints of the two strands were consist- ofABC excinuclease is known to bind to damaged DNA ently different in thatUvrA completely protected the bottom preferentially and is therefore assumed to be the recognition (nonadducted) strand while the top (adducted) strand was subunit of the enzyme. (Seeberg and Steinum, 1982; Husain only partially protected at equivalent protein and DNA con- et al., 1985). To investigate the interactions of this subunit centrations. No estimate of the stoichiometry for UvrA spe- with DNA, the effect of UvrA concentration on the DNase I cific binding could be made due to the relatively low ratio of digestion pattern for the HMT-modified and nonmodified specific to nonspecific binding of UvrA. 137-bp DNA fragments was examined. The results of such an The Effect of UvrB Binding on the UvrA Footprint-UvrB experiment aredisplayed in theautoradiograms in Fig. 2. The has little or no binding affinity for DNA in the absence of UvrA protein protected a33-bp region, which is bracketed by UvrA, but binds tightly to the UvrA.DNA binary complex. (Kacinski and Rupp, 1981; Yeung et al., 1983; Husain et al., arrows. For consistency we have marked these borders the 1985; Yeunget al., 1986). Having obtained the UvrA footprint, same in all the figures, although some differences in the length we next wanted to determine what effect the UvrB subunit of the footprints were seen from experiment to experiment, had on the binding of UvrA to theHMT-modified substrate. due to the partial protection observed at the borders of the The results of these experiments are shown in Fig. 3. There footprint. At low concentrations (9.6-96 nM), the UvrA sub- were threeimportant effects of UvrB binding. First, the unit bound specifically to thefuran-side HMT-monoadducted presence of UvrB induced an apparent conformational change DNA in a concentration-dependent manner, while at higher in the UvrAeDNA complex such that binding of the UvrA concentrations (100-300nM) nonspecific binding occurred. and UvrB subunits to thepsoralen-modified DNA caused the The nonspecific binding resulted in a general disappearance 11th phosphodiester bond 5' to the HMT-adducted thymine of DNase I bands in boththe modified and nonmodified DNA (between G,, and &) to become hypersensitive to DNase I (band IV, lanes 6, 7, 13, 14, Fig. 2A). UvrA binding was cleavage (Band ZZI, lanes 8-14, Fig. 3A). Second, this hyper- analyzed quantitatively by densitometric scanning of partic- sensitivity was accompanied by an apparent decrease in the ular bands (marked by Roman numerals, see Fig. 2 legend) size of the UvrA footprint from 33 to 19 bp. Third, the addition from appropriately exposed autoradiograms. These UvrA sat- of UvrB increased the affinity of UvrA severalfold for the uration curves were in turn used to determine the equilibrium psoralen-modified DNA; this is depicted by the change in constants for specific and nonspecific binding. The results of saturation curves (Fig. 3C), from which an equilibrium con- such an analysis for the top strand are shown in Fig. 2C. A stant was derived. As with UvrA alone we observed more similar analysis for the bottom strand gave essentially the efficient binding of the UvrA.UvrB complex to the bottom same results (datanot shown), although the values were strand of the psoralen-adducted fragment, and we therefore slightly higher due to the clearer footprint that was consist- report the equilibrium binding constant as arange with K, = ently obtained for the bottom strand of the psoralen-modified 1.2 - 6.7 x 10' M-'. It should be noted, however, that the substrate. We therefore give a range of values for specific and nonspecific binding constants as K. = 0.7 - 1.5 X 10' M-', D. K. Orren and A. Sancar, unpublished results. FootprintExcinuclease of ABC 13183

A TOP STRAND TOP STRAND 1234567891ol1I213l41516

r $3 uvr A - 90 I Uvr A !- -U -m

IP -E

.. I.- "" . "_. < -

" BOTTOM STRAND B 1234567AGTC891011121314

UW

L -I "0 -D -m

E 4

0 4 I? '7 '6 2

This suggests that the active form of the UvrA and UvrB 5c complex contains one UvrBmolecule. Effect of the UvrC Subunit-Addition of the UvrC subunit 6C to the UvrA .UvrB .DNA ternary complex results in DNA incisions at the 8th and the 5th phosphodiester bonds5' and 70'- - 3' (respectively) to the psoralen-modified thymine (Van Hou- @"so. - 71 ten et al., 1986a). Because of these dual incisions, it was not possible to observe an effect of the UvrC subunit on UvrA-the c' ._ 3 UvrB footprint on the damaged (top) strand. Surprisingly, a - "C" Uvr A even on the nondamaged strand, UvrC, over a wide range of 9dSo ---"-- I concentrations, had no detectableeffect on thesize or quality - - .. - " " . __ -- of the DNase I footprint (Fig. 5). UvrC binds to bothdouble and single strand DNA, with the latter typeof binding being """0. moreefficient (Sancar et al., 1981). In DNase-footprinting loo'. experiments UvrC had no effect on the affinity of the UvrA ---. ""- or theUvrA and UvrB subunits to the psoralen-monadducted FIG.5. Effect of UvrC subuniton the UvrA-UvrB footprint. DNase I digestion of 5' terminally labeled bottom strand HMT- DNA (data not shown) and UvrC alone exhibited nospecific modified 137-bp DNA. Lane 1, DNase I alone; lane 2, DNase I plus or nonspecific binding to DNA (Figs. 5 and 6, A, lane 4, and 4.8 nM UvrA, lanes 3-8, DNase I plus 4.8 nM UvrA, 5.3 nM UvrB, B, lane 7). and 0.0, 10, 15, 20, 28, and 49 nM UvrC, respectively. Summary of DNase I Footprints during the Assembly of A BC Excision Nuclease-The representative footprints for tecting a33-bp region in manner which is not dependent the assembly of the active form of the enzyme are shown in upon, but stimulated(5-10-fold) by the presenceof ATP (Fig. Fig. 6. At the concentrationsused in these experiments(2-20 6A, lane 8, on 6B, lane 2, and data not shown). There is a nm), there is little or no bindingof any of the three subunits characteristic decrease in the lengthof the DNase I footprint to theunmodified 137-bp DNA fragment (lanes2-4, Fig. 6A). from 33 to 19 bp when both the UvrA and UvrB subunits Furthermore, when UvrB or UvrC were added separately no bind to the psoralen-modified DNA (Fig. 6, A, lanes 7 and 8 detectable binding was observed (Fig. 6B, lanes 6 + 7, respec- and B, lanes 2 and 3).The addition of all three subunits to tively). the psoralen-modified 137-bp fragment resulted in dualDNA UvrA binds to the psoralen-modified DNA fragment pro- incisions at positions Twiand AT,, and can be seen as two bands marked by the Roman numerals I1 and 111 (Fig. 6A, TOP STRAND lane 10). Note that when the substratewas labeled on the 5' 12345678 terminus of the strand, the band corresponding incision to on the 3' side of the thymine adductcould only be observed if 3' incision occurred in the absence of 5' incision. The ability to discern the 3' incision site on 5"labeled DNA indicates that ABC excinucleaseoccasionally cleaves on the 3' side of a DNA adduct in the absence of a 5' incision. We have found thatboth pH and ionic strength of the buffer cancause uncoupling of the 3' and 5' incisions.:' We also observed a low but detectable level of anomalous 80 DNA incisions in the nonadducted strand (lane 5, Fig. 6, A 0 1Uvr A and B). Longer exposures of these gels revealed that these 7c incisions were consistentwith ABC excinuclease-mediated DNA cleavage of the 8th phosphodiester bond 5' to guanines. JB It is known that the phosphotriester method of oligonucleo- 6C Uvr A tide synthesis can lead to damaged guanines at a low fre- quency, and we therefore concluded that these bands were 1 due to incision of the damaged guaninesby ABC excinuclease. The results of the DNase I-footprinting experiments are 5c shown schematically inFig. 7. The DNA incision sites (shown by heavy arrows) prevented an analysisof the effect of UvrC FIG. 4. Stoichiometricbinding of the UvrB subunitto on the top strand footprint. Analysis of the bottom strand UvrA.DNA binary complexes. 5' terminally labeled top strand footprints did not reveal any differences in the UvrA-UvrB 137-bp HMT-modified DNA (4.4 nM) was digested with DNase I and UvrA-UvrB-UvrC footprints. The results summarized in either alone (lanes I) or in the presence of 96.2 nM UvrA together this figure indicated that ABC excinuclease contacted a rela- with 0.0, 1.3, 1.8, 2.6, 5.3, 10.6, or 25.9 nM UvrB (lanes 2-8). The tively small region around the adducted nucleotide. To sup- addition of UvrB produced a hypersensitive DNase I cleavage site, which resulted in the appearance of a new DNA band which is marked port thisconclusion we conducted the experimentsdescribed by I. The intensity of this band was used as an indicator for UvrB below. binding to the binary UvrA.DNA complex. The region of DNase I Minimal LengthSubstrate-We also studied the interaction protection by the UvrA subunit or the UvrA and UvrB subunits is bracketed by arrows. B. Van Houten and A. Sancar, unpublished results. Footprint of ABC Excinuclease 13185

TOP STRAND

YY). 90. 80' - & FIG.7. Summary of the DNase I footprint of ABC excinu- clease during various stages of assembly. For clarity only the central 55 bp of the 137-bp psoralen-modified DNA fragment are displayed. The psoralen-adducted thymine iscircled. The boxes above and below the sequence indicate the minimum area of protection from DNase I digestion. The heauy arrows indicate the positions of the phosphodiester bonds which are cleaved by ABC excinuclease. The light arrow indicates the position of the phosphodiester bond which becomes hypersensitive to DNase I cleavage upon the forma- tion of the UvrA.UvrB.DNA ternarycomplex.

@OTTOM STRAND ..I

Hae III EcoRI DdeI Hinf I

percent incision: 2.96 015 460 6.30 FIG.8. Efficiency of ABC excinuclease incisions on psora-

" len-modified DNA substrates of various lengths. Terminally " labeled top strand 40-bp HMT-modified DNA was digested with the indicatedrestriction enzymes, andthe fragments containing the HMT-modified thymine were purified on polyacrylamide gels. The purified restriction fragments were digested with ABC excision nu- clease and a portion of these reactions were loaded onto 12% polya- crylamide-sequencing gels. The extent of ABC excinuclease incision Uvr A was determined by scintillation counting of the gel slice containing the appropriate DNA band. The percent incision given under the restriction enzyme indicates the extent of 3' incision by ABC exci- nuclease for a HaeIII-digested fragment and the extentof 5' incision for either the EcoRI; DdeI- or HinfI-digested fragments normalized for the incision observed with ARC excision nuclease on the 40-bp DNA fragment. FIG. 6. DNase I footprints of the UvrA subunit, the UvrA and UvrB subunits, and the complete ABC excinuclease. A, 5' promote productiveDNA binding andallow DNA cleavage to terminally laheled topstrand nonmodified (lanes 1-5) orHMT- occur. Restriction enzyme protection experiments with both modified (lanes 6-10) 13'7-bp DNA were treated with DNase I (lanes the UvrA and UvrB subunits bound to the 40-bp substrate 1-4 and 6-9) in the presence of the subunits of ABC excinuclease. Lunes 1 and 6, DNase I alone; lanes 2 and 7,19nM UvrA; lanes 3 and were consistent with the binding domainsidentified by DNase 8, 19 nM UvrA and 5 nM UvrB; lanes 4, 5, 9, and 10, 19 nM UvrA, 5 I protectionexperiments with the 138-bpDNA fragment. nM UvrB, and 20 nM UvrC. B, 5' terminally labeled bottom strand Namely, the restriction enzymes, HinfI and HaeIII were par- psoralen modified 137-bp DNA was digested with DNase I (lanes 1- tially inhibitedby UvrA and UvrB binding,whereas DdeI was 4, 6, and 7) in the presence of various amounts of the Uvr subunits. completely inhibited (data not shown). The positions of the Lune 1, DNase I alone; lane 2, 19 nM UvrA; lane 3, 19 nM UvrA and restriction enzyme sites are shown in Fig. 8. The HaeIII site 5.3 nM UvrB; lane 4, 19 nM UvrA, 5.3 nM UvrB, and 28 nM UvrC; lane 5, as in lane 4 but in the absence of DNase I digestion; lane 6, is 9-bp 5' and theHinfI site is 17-bp3' to the HMT-adducted 5.3 nM UvrB; lane 7,49 nM UvrC; lane R,S'-labeled top strand HMT- thymine; both sites are outside theregions protected against modified DNA treated as in lane 5. Lanes A, G, T,and C refer to the DNase I digestion by the UvrA and UvrB subunits. By con- Maxam-Gilbert sequencing reactions for A + G, G,T + C, and C, trast the DdeI site is only 14 bp 3' to the modified thymine, respectively. The region of DNase I protection by the UvrA subunit just at the borderof the UvrA-UvrB footprint. EcoRI diges- or the UvrA and UvrB subunits is bracketedby arrows. The addition tions were partially inhibited by the psoralen-thymine adduct of the UvrC subunit to the UvrA and UvrB subunits produceda footprint that was not different from that seen with UvrA and UvrB. at position 20 and were not suitable for analysis. We next wanted to determineif the regions of DNA beyond the areas detectedby DNase I footprinting contributed to the of ABC excinuclease with DNA using the 40-bp substrate. overall stability of the complex formation and could be re- We had engineered several convenient restriction sites into a flected in the extentof DNA incision. The terminally labeled 40-bp DNA fragment for two reasons. First, we wanted to 40-bp substrate was prepared as described under "Materials determine if the binding of the Uvr subunits to themodified and Methods," digested with the appropriate restriction en- DNA fragment would protect the DNA fragmentfrom cleav- zyme andthe top strand-labeled fragment containing the age by a particular restriction enzyme. Secondly, by digesting HMT-modified thyminegel purified. These labeled fragments the 40-bp fragment into shorter length fragments we hoped were then digested with ABC excinuclease and the extentof to delineate the minimal contact siteswhich are necessary to 5' or 3' incision was quantitated by determining the radio- 13186 Footprint of ABC Excinuclease activity in the gel slice containing the intact and the ABC DNA and more so in the presence of damaged DNA (Thomas excinuclease-generated incision bands. As shown in Fig. 8, et al., 1985). The function of ATP hydrolysis is not well removal of 9 bp from the 5' end by HaeIII digestion resulted understood at present. Several proteins, including catabolite in a decrease in the 3' incision efficiency to 3.0%. Similarly, activator protein and RNA polymerase, have been shown to digestion with HinfI, DdeI, or EcoRI reduced the 5' incision undergo a characteristicchange in their interactionwith DNA efficiency to 6.3, 4.6, and 0.15%, respectively. These results upon binding of nucleotide substrates or cofactors (Carpousis suggest that regions of DNA beyond the incision sites greatly and Gralla, 1985; Straney and Crothers, 1985; McClure, 1985; stabilize the formation of the active ABC excision nuclease Liu-Johnson et al., 1986). With these proteins,ligand binding complex. Increasing the length of the substrate from 40 to promotes a shift from a less to a more specific DNA binding 137 bp increased the overall incision efficiency from 30-40% mode for a particular sequence. to 50-60%. The hypersensitive site createdby UvrB binding was noted previously with the 40-bp substrate (Van Houten et al., DISCUSSION 1986a). There the 10th and 11th phosphodiester bonds 5' to We have used DNase I footprinting to study the assembly the psoralen-modified thymine were sensitized to DNase I of ABC excinuclease at the siteof a DNA adduct. The infer- cleavage. The appearance of DNase I hypersensitive sites ences drawn from these results, when viewed in the context have also been observed for the binding of the UvrA and of previously published experiments help to formulate a mech- UvrB subunits to a DNA fragment containing a pyrimidine anism ofhow ABC excinuclease selectively binds toand dimer4 and may be responsible for the unique phenotype of cleaves a DNA substrate containing adamaged nucleotide. the uurC-mutants (Van Houten et al., 1986). The UvrA subunit binds specifically to damaged DNA in The UvrC subunit, at theconcentrations used in this study, an ATP-stimulated reaction (Seeberg andSteinum, 1982) displayed no affinity for the damaged DNA substrate and did protecting a 33-bp region from DNase I digestion with an not affect the UvrA or the UvrA and UvrB binding as deter- equilibrium constant of K, = 0.7 - 1.5 X 10' M-'. An estimate mined by DNase I protection experiments. Previous studies of the nonspecific binding affinity was found to be K, = 0.7 suggested that following the concerted DNA incision the three - 2.9 X IO5 M-' (bp) suchthat theUvrA subunit bound to the subunits stay bound at thesite of the DNA damage; the dual damaged DNA with a specificity ratio of lo3 compared to action of helicase I1 and Pol I are needed to displace the nondamaged DNA. Another aspect of the DNase I experi- subunits and allow them to act catalytically (Husain et al., mentspresented here is that protectionobtained on the 1985; Caron et al., 1985). The footprint data are consistent with the UvrA and UvrB subunits staying bound to the site nonadducted (bottom) strand was consistently clearer than after DNA cleavage, although no conclusion can be reached that observed for the adducted (top) strand. This inequality concerning the fate of the UvrC subunit. inthe footprints might be indicative of a more intimate ABC excinuclease acts on many dissimilar DNA adducts contact of UvrA with the nonadducted strand as compared to with essentially the same incision mechanism. What then is the adducted strand. A similar pattern has been observed for the recognition signal for the specific binding of ABC excision the binding of T7 RNA polymerase to the 410 promoter of nuclease? Pearlman et al., (1985), have suggested that three T7 phage. The binding of T7 RNA polymerase to this pro- common pertubations associated with bulky DNAdamage are moter was shown to protect only the template strand from local DNA unwinding (20-90") helical bending or kinking, digestion by DNase I (Basu and Mitra, 1986). (25-45"), and displacement of the two helical axes relative to It has been shown that UvrB alone does not bind to modi- one another (2.5-3.5 A). Based on the asymmetric incision fied DNA but forms a ternary complex with UvrA and DNA preference of ABC excinuclease for the furan-adducted strand (Kacinski and Rupp, 1981; Yeung et al., 1983; Husain et al., of psoralen-cross-linked DNA as a model, we have previously 1985; Yeung et al., 1986). The addition of the UvrB subunit suggested that ABC excinuclease binds to only one face of the caused the following important changes in themode of Uvr A DNA helix at the damage-induced kink (Van Houten et al., binding: (i) the size of the footprint decreased to 19 bp; (ii) 1986b). This model is further supported by incision studies the binding affinity increased to 1.2 - 6.7 X 10' K'; (iii) a performed on UV-irradiated DNA with ABC excinuclease in DNase I hypersensitive band appeared 11 phosphodiester the presence of DNA photolyase: photolyase binds to the bonds 5' to the modified thymine (Fig. 3, A and B). These damaged strand(Husain et al., 1987) andstimulates the alterations indicate that the UvrA. UvrB .DNA complex is binding of ABC excinuclease (Sancar et al., 1984; Myleset aL, significantly different than the UvrA. DNAcomplex. Al- 1987). The differences in the protection of the adducted and though kinetic experiments suggest that the active complex nonadducted strands by ABC excision nuclease reported here consists of one of each subunit (Husain et al., 1985), hydro- lends further supportto thismodel. Verification of the model dynamic studies2 and DNA unwinding studies (Ohet al., 1986) awaits the determination of more precise contact sites of the with UvrA suggest that its active form may be a dimer at the Uvr subunits on damaged DNA by conducting chemical foot- concentrations of proteins used in these experiments (2-40 printing experiments. nM). The decrease in the size of the UvrA footprint with the addition of UvrB might indicate a displacement of one of the Acknowledgments-We thank Drs. Gwen Sancar, Intisar Husain, UvrA monomers, which is then free to participate in another and Gary Myles for their helpful discussions and critical reading of round of substrate binding. Alternatively, UvrA binding might this manuscript. We also thank Suzanne Cheng for help in prepara- be imprecise, so that in a population of DNA molecules the tion of the short oligonucleotides and Applied Biosystems for their overall footprint might appear larger. In this case addition of gift of the long (30-60-mers) oligonucleotides. UvrB serves to promote binding of UvrA in a precise manner. REFERENCES Yeung et al., (1986) have shown that the addition of UvrB Arikan, E., Kulkarini, M. S., Thomas, D. C., and Sancar, A. (1986) stabilizes the binding of UvrA to damaged substrate. 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