Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6774-6778, October 1985 Biochemistry Effect of DNA I and DNA 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 removes 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 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- . 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 on a multicopy plasmid (18). The detail of this containing modified . 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 were obtained UvrC (Mr, 66,038). The three proteins acting in concert from Boehringer Mannheim, and E. coli DNA and T4 hydrolyze the eighth 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. coli in vivo. detailed form. The curve in A of the left panel was superimposed in Stimulation of the Incision Activity of UvrABC Excision B through D (broken line) for comparison. The reactions were carried Nuclease. The reaction kinetics of incision of UV-damaged out in 140 Al of excision repair buffer which contained 0.313 pmol of pBR322 carrying 10 photoproducts (3.13 pmol of adducts), 0.4 pmol DNA by UvrABC excision nuclease in the presence or of UvrA, 0.64 pmol of UvrB, 1.65 pmol of UvrC, and 30 pmol of pol absence ofthe other components ofexcision repair are shown I, 2.8 pmol of helicase, and 220 pmol of T4 DNA ligase when in Fig. 2. From this figure the following conclusions can be indicated. The reactions were started by addition of UvrC protein. made. (i) UvrABC excision nuclease turns over very slowly, Samples (10 Al) were taken after 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, if at all. (ii) pol I or helicase II by themselves cause a slight and 25 min (lanes 1-14) ofincubation at 370C. The reaction was stopped by adding NaDodSO4 to the samples to 0.5%. UvrABC excision nuclease produced 0.01 cut per min andT4ligase produced 0.008 cut per < Cn Um min on nonirradiated DNA; these background values were subtracted > 0 . :)t D D: 0Lo i i Mr XxIO3-3 to obtain the specific cutting values plotted in the figure.

-97 turnover of the nuclease. (iii) In the reaction mixture con- taining all the known excision repair proteins, the initial rate _ - -66 of incision is the same as that obtained by UvrABC excision nuclease alone (Fig. 2D, Inset), suggesting that pol I and -45 helicase II do not assist the excision nuclease in substrate recognition and nucleolytic activity. (iv) The combination of -36 pol I, helicase II, and DNA ligase causes biphasic reaction -29 kinetics. We take the rate beyond the point where incision by ABC nuclease alone reaches a plateau (Fig. 2A) as indicative _ -20 ofthe turnover ofthe excision nuclease. By taking the plateau as a measure of active enzyme molecules, we then obtain a turnover rate ofabout 0.08 cuts per min per UvrABC excision FIG. 1. Complete set of nucleotide excision repair proteins. that the contains one of Approximately 0.5 ,Ag of each protein used in our excision repair nuclease complex (assuming complex reconstitution experiments was electrophoresed on a NaDod- each of the subunits). In Table 1 we present data that show S04/polyacrylamide gel which was stained with Coomassie blue. Hel that the effect ofhelicase II is specific and cannot be replaced II, UvrD protein (helicase II); Lig, T4 DNA ligase. by E. coli Rep protein. Furthermore, the stimulatory effect of Downloaded by guest on October 1, 2021 6776 Biochemistry: Husain et al. Proc. Natl. Acad. Sci. USA 82 (1985) helicase II is completely abolished by anti-helicase II anti- bodies. It is also clear that the stimulatory effect of pol I and -LJ helicase II is not dependent on the ligation step of excision (-) 6

repair, but is partially dependent on the polymerization step -j because a lower level of stimulation was noted when dNTPs 05 were omitted from the reaction mixture. However, in the absence of ligase and dNTPs extensive nick-translation or exonucleolytic degradation of the DNA by pol I takes place 0 making comparison with the excision and filter binding data difficult. Therefore, all our incision assays were carried out in 2_ the complete nucleotide-excision-repair buffer so that results from all three types of assays could be directly compared. It is interesting to note that phage T4 DNA polymerase and the Klenow fragment can replace pol I in the complete system but only in the presence of all four dNTPs. 3 6 9 1 2 15 18 21 25 45 Stinulation of the Excision Activity of UvrABC Excision TIME (min) The pol I II was Nuclease. effect of plus helicase also FIG. 3. Effect of pol I and helicase II on removal of cisplatin investigated using the excision assay, which measures the adducts from DNA. The reactions were carried out in 175-p.l excision removal of the modified nucleotides from DNA. For this repair buffer containing 2.21 pmol of pBR322 (31 pmol of adducts), assay we used [3H]cisplatin-modified DNA and determined 5.6 pmol of UvrA, 8 pmol of UvrB, and 8 pmol of UvrC proteins, the kinetics of the removal of label by UvrABC excision respectively. When indicated DNA polymerase (700 pmol), helicase nuclease under conditions similar to those used for the 11 (9 pmol), and T4 ligase (603 pmol) were also included. Samples incision assay. In Fig. 3 we present the results of these were withdrawn at the indicated time points, the reaction was experiments. In agreement with those obtained by the inci- stopped with 0.5% NaDodSO4 and the amounts of adducts removed sion assay the pol I plus helicase II combination increases the were determined. UvrABC excision nuclease alone (o); the complete extent of the reaction but not the initial rate, thus excision repair system (A). In this assay there was no background strength- removal of cisplatin adducts by any of the proteins used. ening the argument that polymerase and helicase do not contribute to lesion recognition and removal but rather to the turnover of the excision nuclease. In this assay as well, the and -C proteins used in the incision assay were <10% those individual proteins had only a marginal effect on the final used in the excision assay. The higher protein concentrations extent of the reaction, which was comparable to that ob- used in the excision assay may favor complex formation and served in the incision assay (data not shown). The turnover thus increase the efficiency of the enzyme. rate obtained from the excision assay, as measured by the Release of UvrABC Excision Nuclease from Substrate. A new rate of excision effective after 9 min of reaction, in the direct method for determining the dissociation of UvrABC complete system is about 0.08 adduct per min per enzyme excision nuclease from DNA (turnover) by pol I plus helicase complex, in excellent agreement with the value of 0.08 II is provided by the nitrocellulose filter binding assay. UvrA obtained from the incision assay. However, in the reaction and UvrB proteins make a very stable complex with UV- mixture containing only UvrABC excision nuclease we irradiated DNA (t112 =55 min; ref. 6). We observe that the observe stoichiometric removal of adducts, whereas in the addition of UvrC protein results in incision on both sides of incision assay the number of cuts has a stoichiometry <1. A the photoproducts but has no significant effect on the likely explanation is that the concentrations of the UvrA, -B, dissociation of the complex. The kinetics of dissociation of

Table 1. Effect of various proteins on UvrABC excision nuclease Additions and subtractions Incisions per plasmid* Activity, % Excision nuclease 0.97 ± 0.15 100 + Pol I + T4 Lig 1.24 ± 0.16 120 + Hel II + T4 Lig 1.15 ± 0.04 116 + T4 Lig 0.98 ± 0.14 100 + Pol I + Hel II 2.60 ± 0.37 268 + Pol I + Hel II + T4 Lig 2.62 ± 0.45 270 + Pol I + Hel II + T4 Lig - dNTPs 1.74 ± 0.50 180 + Pol I + Hel II + T4 Ligt 2.63 ± 0.16 270 + Pol I + Hel II + E. coli Lig 2.70 ± 0.42 279 + Pol I + Rep protein + T4 Lig 1.14 ± 0.17 118 + Pol I + Hel II + T4 Lig + anti-helicase II 0.74 ± 0.05 77 + T4 Pol + Hel II + T4 Lig 2.30 ± 0.37 238 + T4 Pol + Hel II + T4 Lig - dNTPs 1.12 ± 0.20 116 + Klenow Frag + Hel II + T4 Lig 2.20 ± 0.23 228 + Klenow Frag + Hel II + T4 Lig - dNTPs 1.29 ± 0.17 134 Data were obtained by the incision assay and activities are expressed relative to the incision level obtained by the UvrABC excision nuclease alone after 25 min incubation at 370C. The reaction conditions were exactly as described in Fig. 2 with the following modifications: the reaction mixture with E. coli ligase (E. coli Lig) contained 25 ,uM NAD; the reaction mixture with Rep protein contained 3 pmol ofenzyme, and the reaction mixture with anti-helicase antibodies contained antibodies in 50-fold molar excess over helicase. T4 DNA ligase, T4 Lig; helicase II, Hel II; Klenow fragment, Klenow Frag; T4 DNA polymerase, T4 Pol. *Data are the mean of 3-7 separate determinations mean ± SEM. tPol I and DNA ligase were used at 1/10th the concentrations as described in Fig. 2. Downloaded by guest on October 1, 2021 Biochemistry: Husain et al. Proc. Natl. Acad. Sci. USA 82 (1985) 6777

the excision nuclease from UV-damaged DNA under various acting together to facilitate the turnover of UvrABC excision conditions are shown in Fig. 4. The complex, in the absence nuclease. of other proteins, is very stable-only H10% of the complex- es are dissociated after 25 min. The addition of pol I or DISCUSSION helicase II destabilizes the complexes somewhat, but the most dramatic effect is observed when both proteins are The main conclusions ofthis work and the reinterpretation of present in the reaction mixture simultaneously. Under these previous in vivo data on excision repair are summarized conditions the initial part of the dissociation curve gives a below. half-life of about 10 min. While a rigorous analysis of these (i) UvrABC excision nuclease does not appear to turn over data requires that the binding efficiency of excision nucle- at a physiological rate in vitro in the absence ofotherfactors. Pol ase-DNA complexes be known and that both the photoprod- I or E. coli DNA helicase II alone have a marginal effect on the ucts and the enzymes are turnover of the enzyme while the combination of the two distributed on the DNA according drastically increases the turnover rate to 0.07-0.08 min- as to a Poisson distribution, an approximate first-order rate measured on two different substrates (pyrimidine dimers and constant can be obtained by assuming a simple first-order cisplatin adducts) by the incision, excision, and filter binding dissociation (25). Using this simplification we obtain the assays. This value is in agreement with the in vivo turnover rate dissociation rate constant, k = 0.07 min', which is in obtained by Tang and Patrick (5) who measured the kinetics of excellent agreement with the turnover rates we obtained from pyrimidine dimer removal in cells kept in buffer following UV the other two assays. However, after about 10 min ofreaction irradiation (liquid holding recovery). Under these conditions it time the dissociation rate slows considerably and, after 45 was found that an E. coli cell could excise about 100 dimers per min of incubation, about 40% of the DNA is retained on the hour. Assuming that there are 10-20 UvrABC excision nuclease filter (data not shown). At present we cannot tell whether this complexes per cell (2-4), the turnover rate for the enzyme is change is a reflection of the fact that UvrABC excision 0.12-0.24 min-, which is in good agreement with the value of nuclease-DNA complexes are heterogeneous and that some 0.07-0.08 min' we obtain in vitro. of the complexes are long-lived or whether it is an artifact of (ii) The stimulation of the excision nuclease by pol I plus the experimental conditions we use to measure the dissocia- helicase II is the result of an increase in the turnover rate ofthe tion. Nevertheless, it is quite clear that the combination ofpol enzyme. These two proteins do not effect the initial rate of I plus helicase II causes a rapid dissociation of at least halfof excision by UvrABC excision nuclease but cause its release the complexes, thus enforcing our conclusion from the from the excision site and lead to its turnover. This effect differs incision and excision assays that these two enzymes are from that which we have reported (19) with E. coli DNA photolyase in that photolyase seemingly affects both the initial rate and the final extent of the excision reaction. 100 0 0 (iii) A number of in vivo studies have shown that, although 0 incision of chromosomal DNA appears normal, excision of 0~~~~~ pyrimidine dimers is reduced in both PoIA- (7) and UvrD- 0~~~~~~ (8-10) . These observations could be interpreted by the old excision repair model (in which incision and excision are U 0 a -~ separate steps), with the products ofpolA and uvrD acting at the D 50 excision step. The discovery that ABC nuclease is an excision 0o nuclease (1) requires that a different explanation be made. We suggest the following explanation: in poLA- or uvrD- mutants UvrABC excision nuclease makes stoichiometric numbers of cuts and the corresponding number of dimers are removed, but 0 the enzyme turns over slowly due to defective pol I or helicase (' IO 1 II. As a result the incisions persist in these mutants for longer 00 10 20 periods giving the appearance of normal incision but defective TIME (min) excision. In wild-type cells the enzyme turns over and the FIG. 4. Effects ofpol I and helicase II on dissociation of UvrABC incisions are closed rapidly. Therefore, at a given moment excision nuclease from DNA as measured by the nitrocellulose filter following UV irradiation both wild-type cells and poLA- or binding assay. The 440-1dl reaction mixture contained 0.22 pmol of uvrDF cells may have the same number of single-strand UV-irradiated radiolabeled pBR322 (2.2 pmol photoproducts) and 1 breaks, even though more photoproducts have been excised in pmol of UvrA, 1.2 pmol of UvrB, and 1.5 pmol of UvrC proteins. wild-type cells than in the mutants. Where indicated pol I (30 pmol), helicase II (5.6 pmol), and T4 DNA (iv) The relatively close correspondence we find with our ligase (220 pmol) were also included. The DNA was mixed with the in vitro rate of excision repair and the in vivo rate is strongly Uvr proteins and incubated at 370C for 5 min, then 1.65 pmol suggestive that UvrA, -B, -C proteins, pol I, helicase II, and unlabeled UV-irradiated DNA was added (17.6 pmol adducts) and DNA ligase are necessary and out incubation was continued for another 5 min at which time the other sufficient for carrying proteins were added (time zero). Samples (50 IA) were taken at the excision repair in its entirety in vivo. In Fig. 5 we present a indicated times and collected by filtration through nitrocellulose model of our current view of nucleotide excision repair. filters, which were then washed with 3 ml of reaction buffer, dried, However, it must be pointed out that some of these proteins and radioactivity measured in a liquid scintillation counter. The might be substituted or aided by other proteins in vivo under specific UvrABC excision nuclease-DNA complexes are defined as certain circumstances. Thus DNA polymerase mutants are those remaining 5 min after addition ofcompetitive DNA as we have not as UV sensitive as Uvr- mutants indicating that DNA found UvrA-DNA or UvrC-DNA complexes are completely re- II and III can partially take on the functions of leased at the end of this period (data not shown). In the experiment pol I. Similarly, recent in vivo (15, 26) and in vitro (19) data shown in this graph the specific complexes retained 40%o of input suggest that photolyase stimulates the removal of DNA on the filter, this amount is taken as 100%1 retention at time pyrimidine zero, and the other time points are reported as the percentage of this dimers by the excision nuclease. However, since the effect of amount. T,UvrABC excision nuclease; *, excision nuclease, photolyase is specific for pyrimidine dimers, the enzyme helicase II, and ligase; o, UvrABC excision nuclease, pol I, and assembly consisting of UvrA, UvrB, UvrC proteins, pol I, ligase; *, UvrABC excision nuclease, pol I, helicase II, and ligase. helicase II, and DNA ligase must be the major excision repair Data shown is representative of at least three separate experiments. system operating in E. coli. In this regard it is worth Downloaded by guest on October 1, 2021 6778 Biochemistry: Husain et al. Proc. Natl. Acad. Sci. USA 82 (1985) /AA 31 1 1 1 1 1 1 IIIWITM 51 ADP+

ATP

5. 1 1111111 flllll1111 3'. 5, ° LA AB/ /P 5 [01 Helicase II

H i 3

5vvn -i-rrS Pol I + Helicase

igase

5 3'

FIG. 5. Model for nucleotide excision repair in E. coli. The UvrA protein is an ATPase which scans DNA and stops momentarily when it encounters a nucleotide adduct. The UvrB protein binds to the UvrA-DNA complex and induces a significant conformational change in the UvrA protein such that the latter subunit's ATPase activity is stimulated 2-3-fold (16). This interaction produces a stable complex at the modified nucleotide site. UvrC protein binds to the UvrA-UvrB-DNA ternary complex to constitute UvrABC excision nuclease, which cuts the eighth phosphodiester bond 5' and the fourth (or fifth) phosphodiester bond 3' to the mono- or dinucleotide adduct. After incision, UvrC protein may be released by UvrD; however, the other two subunits remain at the cutting site. The UvrA and UvrB proteins, together with the excised oligonucleotide, are displaced by the combined and specific action of pol I and helicase II. The excision gap is filled in by the polymerase as the displacement occurs and ligase seals the resulting nick.

mentioning that liquid holding recovery does not occur in 10. Kuemmerle, N. B. & Masker, W. E. (1980) J. Bacteriol. 142, uvrD mutants (27), suggesting that helicase II cannot be 535-546. replaced by other DNA . 11. Kuemmerle, N. B. & Masker, W. E. (1983) Nucleic Acids Res. 11, 2193-2204. 12. Kumura, K., Sekiguchi, M., Steinum, A.-L. & Seeberg, E. Note: While this work was in progress we learned that Dr. Lawrence (1985) Nucleic Acids Res. 13, 1483-1492. Grossman and co-workers have obtained results similar to ours. We 13. Setlow, R. B. & Carrier, W. L. (1964) Proc. Natl. Acad. Sci. thank Dr. Grossman for informing us of their conclusions prior to USA 51, 226-231. publication. 14. Boyce, R. P. & Howard-Flanders, P. (1964) Proc. Natl. Acad. Sci. USA 51, 293-300. 15. Hays, J. B., Martin, S. J. & Bhatia, K. (1985) J. Bacteriol. We thank Drs. G. Sancar, M. Caplow, and C. Carter for useful 161, 602-608. discussions. This work was supported by the National Institutes of 16. Thomas, D. C., Levy, M. & Sancar, A. (1985) J. Biol. Chem. Health Grant GM 32833 and by a Presidential Young Investigator 260, 9875-9883. Award from the National Science Foundation to A.S. 17. Abdel-Monem, M., Chanal, M. C. & Hoffmann-Berling, H. (1977) Eur. J. Biochem. 79, 33-38. 1. Sancar, A. & Rupp, W. D. (1983) Cell 33, 249-260. 18. Toucher-Scholz, G. & Hoffmann-Berling, H. (1983) Eur. J. 2. Sancar, A., Wharton, R. P., Seltzer, S., Kacinski, B. M., Biochem. 137, 573-580. Clarke, N. D. & Rupp, W. D. (1981) J. Mol. Biol. 148, 45-62. 19. Sancar, A., Franklin, K. A. & Sancar, G. B. (1984) Proc. Nati. Acad. Sci. USA 81, 7397-7401. 3. Sancar, A., Clarke, N. D., Griswold, J., Kennedy, W. J. & 20. Husain, I., Chaney, S. G. & Sancar, A. (1985) J. Bacteriol. Rupp, W. D. (1981) J. Mol. Biol. 148, 63-76. 163, 817-823. 4. Yoakum, G. H. & Grossman, L. (1981) Nature (London) 292, 21. Riggs, A. D., Bourgeois, S., Newby, R. F. & Cohn, M. (1968) 171-173. J. Mol. Biol. 34, 365-368. 5. Tang, M.-S. & Patrick, M. H. (1977) Photochem. Photobiol. 22. Oeda, K., Horiuchi, T. & Sekiguchi, M. (1982) Nature (Lon- 26, 247-255. don) 298, 98-100. 6. Yeung, A. T., Mattes, W. B., Oh, E. Y. & Grossman, L. 23. Maples, V. R. & Kushner, S. R. (1982) Proc. Nati. Acad. Sci. (1983) Proc. Natl. Acad. Sci. USA 80, 6157-6161. USA 79, 5616-5620. 7. Boyle, J. M., Paterson, M. C. & Setlow, R. B. (1970) Nature 24. Hickson, I. D., Arthur, H. M., Bramhill, D. B. & Emmerson, (London) 226, 708-710. P. T. (1983) Mol. Gen. Genet. 190, 265-270. 25. Madden, J. J. & Werbin, H. (1974) Biochemistry 13, 8. van Sltjis, C. A., Mattern, I. E. & Paterson, M. C. (1974) 2149-2154. Mutat. Res. 25, 273-279. 26. Yamamoto, K., Satake, M. & Shinagawa, H. (1984) Mutat. 9. Rothman, R. H. & Clark, A. J. (1977) Mol. Gen. Genet. 155, Res. 131, 11-18. 267-277. 27. Tang, M.-S. & Smith, K. C. (1981) Mutat. Res. 80, 15-25. Downloaded by guest on October 1, 2021