Escherichia Coli DNA Photolyase Stimulates Uvrabc Excision Nuclease in Vitro (Pyrimidine Dimers/DNA Repair/DNA-Protein Interactions) Aziz SANCAR, KATHY A

Home , Lyase, Photolyase

Proc. NatI. Acad. Sci. USA Vol. 81, pp. 7397-7401, December 1984 Biochemistry Escherichia coli DNA photolyase stimulates uvrABC excision nuclease in vitro (pyrimidine dimers/DNA repair/DNA-protein interactions) Aziz SANCAR, KATHY A. FRANKLIN, AND GWENDOLYN B. SANCAR University of North Carolina School of Medicine, Department of Biochemistry, Chapel Hill, NC 27514 Communicated by Mary Ellen Jones, August 13, 1984

ABSTRACT Pyrimidine dimers are the major photoprod- clease have different action mechanisms and substrate speci- ucts produced in cellular DNA upon UV irradiation. In Esche- ficities suggests that they interact with pyrimidine dimers richia coli there are dark and photorepair mechanisms that differently. However, it is conceivable that binding of one eliminate the dimers from DNA and prevent their lethal and enzyme to the dimer substrate may interfere with binding of mutagenic effects. To determine whether these repair mecha- the other. Such a situation becomes of physiological signifi- nisms act cooperatively or competitively in repairing DNA, we cance when photolyase binds to pyrimidine dimers but can- investigated the effects upon one another of DNA photolyase, not repair them for lack of photoreactivating light. Under which mediates photorepair, and uvrABC excision nuclease, such conditions, does the enzyme impede DNA repair by an enzyme complex of the uvrABC gene products, which cata- uvrABC excision nuclease? While in vivo data (10, 11) have lyzes nucleotide excision repair. We found that photolyase suggested that photolyase in fact aids dark repair, it has been stimulates the removal of pyrimidine dimers but not other reported (12) that in vitro the enzyme inhibits uvrABC exci- DNA adducts by uvrABC excision nuclease. The two subunits sion nuclease. Having recently purified both enzymes to ho- of uvrABC excision nuclease, the uvrA and uvrB proteins mogeneity (8, 13), we decided to study their effects on one which together bind to the dimer region of DNA, had no effect another. In this communication we demonstrate that photo- on the activity of photolyase. T4 endonuclease V, which like lyase does in fact stimulate the specific removal of pyrimi- photolyase is specific for pyrimidine dimers, was inhibited by dine dimers by uvrABC excision nuclease, thus strongly sug- photolyase, suggesting that these two proteins recognize the gesting that, even in the dark, the enzyme contributes to the same or similar chemical structures in UV-irradiated DNA repair of UV-damaged DNA. that are different from those recognized by uvrABC excision nuclease. MATERIALS AND METHODS Bacterial Strains. E. coli K-12 derivatives CSR603 (recAl In Escherichia coli UV (200-300 nm)-induced DNA damage uvrA6 phr-J) and AB2487 (recAl thyA) were obtained from is repaired by three molecular mechanisms: photoreactiva- B. J. Bachmann and P. Howard-Flanders, respectively. tion (1), nucleotide excision repair (2, 3), and postreplica- Enzymes. The subunits of uvrABC excision nuclease were tion-recombination repair (4). Of these repair mechanisms, prepared essentially as described by Sancar and Rupp (8) the postreplication-recombination pathway does not involve with the exception of uvrB protein, for which a strain carry- an enzymatic machinery that recognizes the photoproducts ing the tac-uvrB plasmid pUNC211 (in which uvrB protein and removes them; rather, it operates on the gaps generated constitutes about 15% of total cellular proteins upon induc- by the replicative mechanism opposite dimers to initiate a tion) was used as the starting material. E. coli DNA photo- recombinational process that ultimately leads to "diluting lyase was purified as described (13). T4 endonuclease V was out" of the photoproducts. On the other hand, photoreacti- a generous gift from E. C. Friedberg and R. A. Schultz. vation and nucleotide excision repair involve enzymes that DNA Purification. Nonradioactive pBR322 plasmid was specifically recognize the DNA damage and directly elimi- purified from AB2487/pBR322 by Sarkosyl lysis, followed nate the photoproducts from DNA. Photoreactivation is me- by centrifugation in CsCl/ethidium bromide density gradi- diated by DNA photolyase, a flavoprotein (5) that acts spe- ents. [3H]pBR322 was prepared as follows: AB2487/pBR322 cifically on the major UV photoproduct, the cis-syn pyrimi- was grown in K medium containing thymidine (2.5 ,ug/ml) to dine dimer, and catalyzes its photolysis back to pyrimidines A6N = 0.6, at which time chloramphenicol was added to 200 by 300- to 500-nm light (6, 7). Nucleotide excision repair is ,ug/ml and growth was continued for another hour. At this initiated by uvrABC excision nuclease (an enzyme complex point [3Hjthymidine (78.1 Ci/mmol, New England Nuclear; of the uvrABC gene products), which cuts the eighth phos- 1 Ci = 37 GBq) was added to a final concentration of 2 phodiester bond 5' and the fourth or fifth phosphodiester ,uCi/ml, and incubation was continued for 14 hr. The cells bond 3' to a pyrimidine dimer to generate a 12- to 13-nucleo- were collected, and the plasmid DNA was purified as de- tide-long single-stranded DNA carrying the pyrimidine di- scribed above. The specific activity of the plasmid was 3.98 mer. The oligonucleotide is removed, and the resulting gap is x 104 cpm/,ug. The plasmid concentration was determined filled by DNA polymerases and sealed by ligase (8). While by using an extinction coefficient of 20 M-1 cm-1 at 260 nm photolyase is specific for pyrimidine dimers, uvrABC exci- and a Mr of 2.88 x 106. sion nuclease removes, in addition to pyrimidine dimers, the DNA Damaging Treatments. UV-damaged pBR322 was second major UV photoproduct, 6-[4'-(pyrimidine-2'-one)J- obtained by irradiating the DNA at 10 jig/ml with a General thymine [Thy(6-4)Pyo] (8, 9) as well as other nucleotide ad- Electric lamp at a rate of 50 ,uW/cm2 for 100 sec in 10 mM ducts generated by such diverse chemicals as psoralens, cis- Tris chloride, pH 7.4/10 mM NaCl/1 mM EDTA (buffer A). diamminedichloroplatinum(II), nitrous acid, and mitomycin This treatment resulted in 1.7-1.8 lethal hits per molecule as C. Thus, the fact that photolyase and uvrABC excision nu- measured by transformation (14). Of the lethal lesions, 95% were photoreactivable and, therefore, were assumed to be pyrimidine dimers. cis-Pt(NH3)2C12-damaged DNA was pre- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: Thy(6-4)Pyo, 6-[4'-(pyrimidine-2'-one)]thymine. 7397 Downloaded by guest on September 29, 2021 7398 Biochemistry: Sancar et aL Proc. Natl. Acad. Sci. USA 81 (1984) pared by incubating pBR322 at 100 ,g/ml with cis- Table 1. Determination of the number of photolyase-pyrimidine Pt(NH3)2CI2 (0.1 mM) at 23°C in buffer A for 35 min and then dimer complexes dialyzing the mixture for 24 hr against the same buffer. This Lethal Dimers Fraction treatment produced on the average 1.43 lethal hits per mole- Surviving hits per per of dimers cule as measured by transformation. Treatment fraction molecule molecule repaired Enzyme Assays. The uvrABC excision nuclease buffer (175 ,1u) contained 50 mM Tris chloride (pH 7.5), 100 mM None 1.0 KCl, 10 mM MgCl2, 10 mM dithiothreitol, 2 mM ATP, 0.57 UV(50 J/m2) 0.170 1.77 1.71 pmol of [3H]pBR322, and uvrA, -B, and -C subunits at the UV + flash 0.55 0.59 0.57 0.66 amounts indicated in Results. The reaction mixture was in- UV + black light 0.90 0.10 1.0 cubated at 23°C; 10-,ul samples were taken at time intervals pBR322 DNA was irradiated at 254 nm with a fluence of 50 J/m2 and mixed with an equal volume of a solution containing 50 at a concentration of 10 ,ug/ml in buffer A and then mixed with mM Tris chloride (8.0), 20 mM EDTA, 1% NaDodSO4, 10% photolyase in uvrABC excision nuclease buffer. The reaction mix- glycerol, and 0.05% bromophenol blue. The samples were ture (555 1.l) contained 0.9 pmol of DNA and 27 pmol of enzyme. loaded onto a 1% agarose gel, which was run and stained After a 10-min incubation in the dark at 230C, the mixture was with ethidium bromide by standard procedures. The bands exposed to a single flash and a sample was taken. The rest of the circu- mixture was photoreactivated for 30 min with black light at a rate of corresponding to covalently closed circular and open 2 W/cm2. The number of lethal hits per molecule at various stages lar DNA were cut out, dissolved in 1 ml of 1 M HC104, and of the experiment were determined by the method of Sancar and neutralized with NaOH; then 10 ml of scintillant was added, Rupert (14), and the number of dimers per molecule was calculated and the radioactivity was quantitated in an LKB liquid scin- by assuming that the lesions remaining after 30 min of photo- tillation counter. From the fraction of covalently closed cir- reactivation were nondimer photoproducts. Recent experiments cular DNA remaining at each time point, the number of cuts (unpublished data) show that photolyase does not act on Thy(6- made by uvrABC excision nuclease was calculated by using 4)Pyo, which probably accounts for most of the "nonphotoreactiva- the Poisson distribution. ble photoproducts." Photolyase was assayed by the E. coli transformation assay as described (13, 15) with the following modifications. ing light flash of about 1-msec duration (16). The results (Ta- The reaction was conducted in 115 ,ul of uvrABC excision ble 1) show that this treatment resulted in photolysis of 66% nuclease buffer containing 0.9 pmol of pBR322 and 0.05 of the dimers. Because in this photolyase preparation only pmol of photolyase. The mixture was illuminated with a 70% of the molecules contain the flavin chromophore (5, 17) General Electric black light at a rate of 2 mW/cm2; 5-,l sam- necessary for photolysis, we conclude that the fraction of ples were taken at time intervals and mixed with an equal dimers bound by photolyase was 66/70 = 0.94. This calcula- volume of 20 mM EDTA and then used to transform compe- tion assumes that photolyase molecules with or without the tent CSR603 cells. chromophore are equally efficient in binding to dimers. This T4 endonuclease V assay was conducted at 37°C in 20 ,ul assumption was confirmed by nitrocellulose filter-binding of buffer A containing 0.07 pmol of pBR322 and the amount experiments in which it was found that =100% of the dimers of enzyme indicated in Results. were complexed with the enzyme under similar experimental conditions (unpublished data). RESULTS Having established the conditions such that, at any given Effect of Photolyase on uvrABC Excision Nuclease. To de- moment, most of the dimers are bound to photolyase, we termine the effect of photolyase on excision of pyrimidine then treated the UV-irradiated DNA with uvrABC excision dimers by the excision nuclease, we wanted to use experi- nuclease in the presence or absence of photolyase and fol- mental conditions such that, at the time of addition of the lowed the cutting kinetics by this enzyme (Figs. 1 and 2). nuclease, most or all of the pyrimidine dimers were bound to The excision nuclease had no effect on unirradiated DNA in photolyase. This was achieved by preincubating UV-irradi- the presence or absence of photolyase. With irradiated DNA ated pBR322 DNA with about a 16-fold molar excess of pho- the enzyme converted covalently closed circles to open cir- tolyase over pyrimidine dimers. To find out what fraction of cles by incising on both sides of the photoproducts (8), and pyrimidine dimers was bound to photolyase under these con- this conversion was stimulated by photolyase, as can be seen ditions, the mixture was exposed to a single photoreactivat- qualitatively in Fig. 1. When the data from Fig. 1 and two No UV UV

1 16 1 16

-J

FIG. 1. Cutting of UV-irradiated pBR322 DNA by uvrABC excision nuclease (ABC) in the presence (Lower) or absence (Upper) of photolyase (PL) as assayed by agarose gel electrophoresis. The reaction mixture (175 ,ul) contained 0.57 pmol of [3H]DNA and 0.52, 1.2, and 2.7 pmol of uvrA, uvrB, and uvrC proteins, respectively, and 14.8 pmol of PL when indicated. The irradiated DNA contained 1.7 photoproducts per molecule, and 1.6 of these were photoreactivable and presumed to be pyrimidine dimers. In the reactions with photolyase, the DNA was preincubated with the enzyme for 10 min at 23°C before the addition of uvrABC excision nuclease. (Left) Unirradiated DNA. (Right) Irradiated DNA. Lanes 1 through 16 contain samples (10 ,ul each) taken at 0, 0.5, 1, 1.5, 2, 2.5, and 3 through 12 min after addition of uvrABC excision nuclease. Downloaded by guest on September 29, 2021 Biochemistry: Sancar et aL Proc. Natl. Acad. Sci. USA 81 (1984) 7399 other experiments conducted under identical conditions were analyzed quantitatively, the results shown in Fig. 2 were obtained. Photolyase stimulated both the initial rate and the extent of the cutting reaction by a factor of -2. Since LUJ 0.8 :D about 95% of the UV lesions in the DNA used in these ex- () LUJ periments were pyrimidine dimers, we conclude that photo- - 0.6 lyase stimulates the removal of pyrimidine dimers by uvr- 0 ABC excision nuclease. Stimulation of uvrABC Excision Nuclease by Photolyase Is LU 0.4 Specific for Pyrimidine Dimers. To understand the mechanism of stimulation by photolyase, it is important to know whether the enzyme stimulates the removal of other DNA adducts that are substrates for uvrABC excision nuclease but not for photolyase. For this purpose we used DNA containing cis-Pt(NH3)2Cl2 or psoralen adducts and conducted 0 2 4 6 8 10 12 the cutting reaction in the presence or absence of photolyase. The results obtained with cis-Pt(NH3)2C12-damaged TIME (min) DNA are shown in Fig. 3. The rate and extent of cutting by FIG. 3. Kinetics of cutting of cis-Pt(NH3)2Cl2-damaged DNA by uvrABC excision nuclease were the same regardless of uvrABC excision nuclease in the presence (A) or absence (o) of pho- whether photolyase was present or absent in the reaction tolyase. The experimental conditions were the same as in Fig. 1 ex- mixture. Similarly no stimulation of cutting of psoralen-dam- cept that the DNA contained 1.45 lethal cis-Pt(NH3)2C12 adducts per aged DNA was observed (data not shown), indicating that pBR322 instead of UV photoproducts. the effect of photolyase on the activity of uvrABC excision nuclease is specific for pyrimidine dimers. dressed the question of whether the binding of these subunits Effect of uvrA and uvrB Proteins on Photolyase. Having has any effect on photolyase. UV-irradiated DNA was prein- established that photolyase stimulates the removal of pyrirni- cubated with a molar excess of uvrA and uvrB proteins and dine dimers by uvrABC excision nuclease, we wanted to then mixed with limiting amounts of photolyase and photo- find out if the effect was reciprocal-i.e., does the excision reactivated (Fig. 4). The same rate and extent of photoreacti- nuclease stimulate the photolysis of pyrimidine dimers by vation were observed, whether uvrA and uvrB proteins were photolyase? Since preincubation of UV-irradiated DNA with present or absent in the reaction mixture, suggesting that the saturating amounts of uvrABC excision nuclease will result binding of these subunits to DNA does not affect the binding in excision of all pyrimidine dimers before the addition of of photolyase. photolyase, we conducted the experiment with only two sub- DNA Photolyase Inhibits T4 Endonuclease V. The results units of the excision nuclease. Data from several labora- presented above suggest that the contact sites of photolyase tories (18-21) suggest that uvrA and uvrB proteins are the and uvrABC excision nuclease on UV-irradiated DNA do subunits involved in specific recognition of and binding to not overlap. Another enzyme that acts on UV-damaged the dimer-containing region of DNA in a reaction driven by DNA is T4 endonuclease V. This enzyme like photolyase, ATP hydrolysis by the uvrA subunit. Therefore, we ad- but unlike uvrABC excision nuclease, repairs only pyrimidine dimers; its action mechanism strongly suggests that, 1.2 when it binds to DNA, it is in close contact with the dimer: 2I0 the enzyme cleaves the N-glycosyl bond of the 5' pyrimidine 1.0 - / of the dimer and then the phosphodiester bond 3' to the LUj apyrimidinic deoxyribose that is generated by the first reac-

08 0 LUJ LU 0 0<- LUj LUJ ur- 0.4 lUJ a) Q. 02 z a 02 o 2 4 6 8 10 12 TIME (min) 0 10 20 30 40 TIME (min) FIG. 2. Kinetics of cutting of UV-irradiated DNA by uvrABC excision nuclease in the presence (A) or absence (o) of photolyase. FIG. 4. Photoreactivation of UV-irradiated DNA in the presence Data from three experiments, including the one shown in Fig. 1, that (A) or absence (o) of uvrA and uvrB proteins. The reaction mixtures were conducted under the same conditions were used to calculate (115 Al) contained 0.9 pmol of pBR322 (1.7 dimers per molecule) and the average number of cuts per pBR322 molecule. The nontreated 0.05 pmol of photolyase in uvrABC excision nuclease buffer; where DNA contained 0.11 cut per molecule, which has been subtracted indicated, the mixture was preincubated for 10 min with 14 pmol of from the time points. Incubation of nonirradiated DNA with uvr- uvrA and 12.8 pmol of uvrB proteins before the addition of photo- ABC excision nuclease with or without photolyase did not introduce lyase. In both cases the samples were kept in the dark for 5 min after additional cuts within the duration of the experiments (see Fig. 1; the addition of photolyase and then exposed to photoreactivating also data not shown). o, DNA treated with ABC nuclease. A, DNA light (2 W/cm2). Samples (5 ,ul) were taken at the indicated times, treated with ABC nuclease after 10 min preincubation with photo- mixed with an equal volume of 20 mM EDTA, and then used for lyase. The average deviation from the mean was + 5% for the three transforming CSR603. The amount of dimers repaired was calculat- experiments. ed by the increase in transformation frequency as described (13, 15). Downloaded by guest on September 29, 2021 7400 Biochemistry: Sancar et aL Proc. Natl. Acad. Sci USA 81 (1984) 1 2 3 be present in the same organism. It is interesting to note that M. luteus lacks photolyase (29), and it is tempting to speculate that other bacteria that lack photolyase (Haemophilus influenzae, Streptococcus pneumonia, Bacillus subtilis) initiate dimer excision by a glycosylase-apyrimidinic acid endonuclease type of a mechanism rather than by an excision nuclease. From the physicochemical point of view, our results re- veal certain aspects of the mechanism of binding of uvrABC excision nuclease and photolyase to dimer-containing DNA. The excision nuclease hydrolyzes the eighth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the dimer (8). Thus, the incision sites are apart by about one turn FIG. 5. Inhibition of T4 endonuclease V by photolyase. The re- of the double helix, suggesting that the enzyme binds to action mixtures (20 1.l) contained 0.07 pmol of pBR322 (irradiated DNA on one face of the helix that does not include the two with 150 J/m2, which produced 4.4 dimers per molecule) in T4 endo- pyrimidines making the dimer; therefore, it does not inter- nuclease V buffer and 7.4 pmol of photolyase and 22 pmol of endo- fere with photolyase, which recognizes the dimer, as op- nuclease where indicated. The reaction mixtures were incubated at posed to uvrABC excision nuclease, which recognizes the 370C for 30 min and then analyzed on an agarose gel. Lanes: 1, irra- helical distortion caused by the dimer (and by other "bulky" diated DNA; 2, DNA treated with endonuclease; 3, irradiated DNA show preincubated with photolyase at 23TC for 10 min before addition of base adducts). Recent experiments (unpublished data) the endonuclease. Quantitative analysis of the gel shows that T4 en- that E. coli photolyase has the same turnover number for donuclease V produced 1.96 and 0.71 nicks per molecule in the ab- dimers in oligo(dT)s containing more than 4 thymidine resi- sence (lane 2) and presence (lane 3) of photolyase, respectively. The dues as for native DNA and that the enzyme repairs dimers linear DNA in lanes 2 and 3 is probably due to a single-strand-specif- in oligo(dT)3 at 43% of maximum rate. Thus, it is clear that ic endonuclease contaminant in the T4 endonuclear V preparation the dimer is the important structure recognized by photo- used. Such an endonuclease would cut across the nicks produced by lyase, and it is reasonable to assume that the enzyme inter- T4 endonuclease V and generate linear molecules. acts with a four-to-six nucleotide region containing the dimer and that these nucleotides do not interact with uvrABC exci- tion (22-26). This type of action mechanism suggests that sion nuclease. Binding of uvrA protein to UV-damaged photolyase and T4 endonuclease V might interfere with each DNA partially unwinds the helix (unpublished data), thus, other's binding to pyrimidine dimers. In Fig. 5 we show the one can speculate that the net effect of the binding of effect of photolyase on cutting by T4 endonuclease V. The UvrA/B to DNA is to increase the single-strandedness cre- enzyme inhibited the endonucleolytic function of the nucle- ated by the dimer. Since photolyase is equally efficient on ase, confirming our expectations that the pyrimidine dimer single- and double-stranded DNA, this increase in single- per se is the important structure for recognition by both en- strandedness is not expected to affect photoreactivation, in zymes. agreement with our observation that uvrA and uvrB proteins did not affect the activity of photolyase. (The effect of uvrC DISCUSSION protein on photolyase was not investigated. It is conceivable that, upon formation of the uvrA/B-DNA complex, uvrC The significance of the results presented in this paper are 2- may have an effect on the binding of photolyase.) On the fold. From the physiological point of view, our findings sug- other hand, since the pyrimidine dimer per se is not the gest that two enzymes that repair pyrimidine dimers in E. structure recognized by the excision nuclease, it is possible coli act in concert to help the survival of the organism. The that photolyase, by binding to the dimer and changing the demonstration in vitro that photolyase stimulates uvrABC DNA helix parameters, makes the DNA a better substrate excision nuclease is in agreement with the in vivo results of for the excision nuclease. Alternatively, photolyase by bind- Harm and Hillebrandt (10), who found that E. coli phr- mu- ing to the dimer may facilitate the dissociation of uvrABC tants were more UV sensitive than phr+ cells even in the excision nuclease from DNA after the incision events; as it dark, and with the results of Yamomoto et al. (11), who has been reported (21) that the nuclease remains complexed found that photolyase-overproducing cells were more UV with DNA for a long period after the incision reaction, such resistant than wild-type cells and that this increased resist- an effect might promote the turnover of this enzyme, thus ance was observed only in Uvr+ cells. On the other hand, increasing the rate and extent of the cutting. Whatever the our results are in contradiction with an earlier report (12) mechanism is, it is clear from our results that the excision that photolyase inhibits uvrABC excision nuclease in vitro. nuclease is stimulated by photolyase specifically in its action However, those studies were conducted with the so-called on pyrimidine dimers. Indeed, it is ironic that uvrABC exci- photolyase R protein, whose identity as a true photolyase sion nuclease is named as such since the major UV photo- and its physiological significance in photoreactivation is product, the pyrimidine dimer, is one of the poorest sub- questionable (13). Our finding of inhibition of T4 endonucle- strates for the enzyme, presumably because the distortion ase V by E. coli photolyase is in accordance with those of caused by the dimer is minimal (30) as compared to that Patrick and Harm (27) that yeast photolyase inhibits Micro- caused by other DNA adducts. In our limited survey of vari- coccus luteus UV endonuclease, an enzyme that has a size ous substrates, we found (unpublished data) the psoralen- similar to that of T4 endonuclease V and the same action pyrimidine mono- and di-adducts to be the best substrates mechanism (28). The inhibition of T4 endonuclease V by followed by Thy(6-4)Pyo, certain cis-Pt(NH3)2C12 adducts, photolyase is probably of little physiological significance in and then pyrimidine dimers. E. coli, as the amount of T4 endonuclease V produced upon infection by'T4 phage is in vast excess over photolyase (26) We thank Michael Levy who purified the uvrC protein used in our and, under such conditions, no significant inhibition is to be experiments and Drs. M. S. Jorns, M. Caplow and C. W. Carter for expected. However, our finding that photolyase inhibits an helpful discussions. This work was supported by the National Insti- enzyme that initiates dimer removal by a glycosylase-apyri- tutes of Health Grants GM 31082 and GM 32833 and by The Presi- midinic acid endonuclease type of mechanism suggests that dential Young Investigator Award from the National Science Foun- such an inhibition might be detrimental should both enzymes dation. Downloaded by guest on September 29, 2021 Biochemistry: Sancar et al. Proc. Natl. Acad. Sci. USA 81 (1984) 7401

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