Proc. Nati. Acad. Sci. USA Vol. 89, pp. 9420-9424, October 1992 Biochemistry Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V (site-directed mutagenesis/DNA repair/UV radiation/oligonudeotide substrate) ToMOKo Doi*t, ACHIM RECKTENWALD*t, YOKo KARAKI*, MASAKAZU KIKUCHI*, KOSUKE MORIKAWA*, MORIO IKEHARA*, TETSUYA INAOKA*¶, NAOKO HoIu§, AND EIKO OHTSUKA§ *Protein Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan; and §Facuity of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan Communicated by Susumu Ohno, May 4, 1992

ABSTRACT T4 endonuclease V [endodeoxyribonucease two activities and the domain structure. As yet, no catalytic (pyrimidine dimer); deoxyribonuclease (pyrimikine dimer), EC mechanism of any PD glycosylase has been elucidated. 3.1.25.1] initiates repafr of damaed DNA by hydrolysis of the The aromatic residue-rich region, Trp-Tyr-Lys-Tyr-Tyr (res- N-glycosyl bond at the 5' side of a pyrimidine photodimer in idues 128-132), near the C terminus of T4 endo V has been double-stranded DNA. To study one of the active sites of T4 implicated as a specific pyrimidine dimer-binding site by the endonudease V, systematic site-directed mutagenesis was per- ability of Lys-Trp-Lys or Lys-Tyr-Lys tripeptides to bind to formed on the synthetic T4 endonuclease V gene, in paraflel with UV-irradiated DNA (5). Furthermore, site-directed mutagene- three-dimensional structure analysis by x-ray crystallography. sis of this region has suggested the importance of aromatic The mutant proteins were evaluated for DNA glycosylase activ- residues in bindingto the pyrimidine dimer(6, 7). In terms ofthe ity using an oligonucleotide duplex (14-mer) containing a single processive binding ability of T4 endo V to DNA, proteins thymidine dimer as a substrate. Replacement of either Glu-23 bearing mutations ofArg-3, -26, or -33 to glutamine were shown with glutamine or asparatic acid or Arg-3 with glutami to have decreased affinity for nontarget DNA (8-10). However, completely abolished DNA glycosylase activity. Mutation of little is known about the catalytic mechanisms of T4 endo V. Arg-3 to lysine or of Arg-26 to glu or lysine in a basic The synthesis ofthe T4 endo V gene and the establishment amino acid cluster caused serious defects in DNA glycosylase of a high-level expression system in Escherichia coli (11) activity, which are reflected in the increases inK. and decreases enabled us to isolate a large quantity ofthe protein, which led in kt of DNA glycosylase activity. On the other hand, substi- to successful crystallization of the enzyme (12). Most re- tutions of lysine for Arg-22 or of glutamine for Arg-117 or cently, the crystal structure has been determined at 1.6A Lys-121 resulted in increases in the Km value. The completely resolution (Fig. 3) (13). The refined structure revealed that T4 inactive mutant proteins, E23Q and R3Q, in which glutamie endo V is classified as an all a protein and is composed of three helices. The aromatic-rich sequence, Trp-Tyr-Lys-Tyr- was substituted for Glu-23 and Arg-3, respectively, were further Tyr (residues 128-132), is located in the C-terminal loop, investigated by CD spectroscopy for their ability to bind the which contributes to the bundling of the three helices. oligonucleotide substrate. It was found that the E23Q protein Moreover, the side chains of basic amino acids, such as retained specific substrate-binding ability, whereas the R3Q Arg-3, Arg-22, Arg-26, Lys-80, Arg-81, Arg-117, Lys-121, protein did not. These results indicate that Glu-23 plays an and so on, form a cluster on the protein surface, in which important role in catalysis of the DNA glycosylase reaction, and Glu-23 is the only acidic residue (see Fig. 7). that Arg-3 is a crucial residue for substrate binding. In addition, To obtain insight into the catalytically active center of T4 Arg-22, Arg-26, Arg-117, and Lys-121 in the basic amino acid endo V, we have performed extensive site-directed mutagen- cluster also participate in substrate binding. We conclude that esis on the synthetic gene and evaluated the PD glycosylase the basic amino acid cluster in T4 endonuclease V is an essential activities ofthe mutant proteins by utilizing an oligonucleotide structure for DNA glycosylase activity. substrate under nonprocessive conditions. Here, we report that the cluster ofbasic amino acid residues plays an important Endonuclease V [endo V; endodeoxyribonuclease (pyrimi- role in specific binding ofT4 endo V to a pyrimidine dimer in dine dimer); deoxyribonuclease (pyrimidine dimer), EC the DNA and that Glu-23, the only acidic residue in the cluster, 3.1.25.1], found in bacteriophage T4, is responsible for ex- is essential for catalysis of the PD glycosylase reaction. cision repair of damaged DNA. The enzyme possesses two activities: a pyrimidine dimer DNA glycosylase (PD glyco- MATERIALS AND METHODS sylase) and an apyrimidinic/apurinic (AP) endonuclease (Fig. 1) (1, 2). The gene (T4 denV) encoding T4 endo V was Materials and Strains. Oligonucleotides were synthesized identified in association with the UV resistance of T4 phage on an Applied Biosystems DNA synthesizer (model 380B). and was shown to encode 138 amino acids (Fig. 2) (3). The Adenosine 5'-[a-[35S]thio]triphosphate and [y-32P]ATP were protein recognizes a pyrimidine photodimer structure in a purchased from Amersham. Restriction enzymes were from double-stranded DNA, hydrolyzes the N-glycosyl bond at New England Biolabs, Boehringer Mannheim, or Takara the 5' side of a pyrimidine dimer, and cleaves the apyrimi- Shuzo (Kyoto). Lysyl endopeptidase was purchased from dinic phosphodiester bond via a (3-elimination reaction. In Wako Pure Chemical Industries (Osaka). addition, T4 endo V is capable of binding and scanning nontarget DNA processively (4). The fact that such a small Abbreviations: endo V, endonuclease V; PD glycosylase, pyrimidine protein possesses two catalytic activities has engendered dimer DNA glycosylase; AP, apurinic/apyrimidinic. considerable interest in terms ofthe relationship between the tTo whom reprint requests should be addressed. tPresent address: Biotechnology Research Institute, National Re- search Council of Canada, 6100 Royalmount Avenue, Montreal, The publication costs of this article were defrayed in part by page charge PQ, H4P 2R2, Canada. payment. This article must therefore be hereby marked "advertisement" Present address: Bio-Organic Research Department, CIBA-Geigy- in accordance with 18 U.S.C. §1734 solely to indicate this fact. Japan Ltd., 10-66, Miyuki-cho, Takarazuka 665, Japan. 9420 Downloaded by guest on October 1, 2021 Biochemistry: Doi et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9421

HN NH HN N N H NH

H H ~~~AP-endmacism 0 0 aHlvity HO CHO HOP.>R RO PO R O (HHO-PO OR 6- HR-~ 6- RO 6- FIG. 1. T4 endo V catalyzes two reactions. T4 endo V recognizes a pyrimidine dimer on double-stranded DNA, hydrolyzes the N-glycosyl bond at the 5' side of a pyrimidine dimer (PD glycosylase activity), and cleaves the phosphodiester bond at the AP sites (AP endonuclease activity). E. coli strains used were HB101 [F-, hsdS20(rj, mj), Assay for PD Glycosylase Activities ofMutant Proteins. The recA13, aral4, proA2, lacY), galK2, rpsL20(Strr), xyl-5, substrate was a duplex of 5' 32P-labeled d(CGAAGGTTG- mtl-i, supE44, leu, mcrB-] for expression of wild-type and GAAGC) and the complementary d(GCTTCCAACCTTCG). mutant proteins and DH1 [F-, recAl, endAl, gyrA96, thi-1, Reactions were carried out in 40 mM Pipes buffer, pH 7.0/0.1 hsdRl7 (rk, mK), supE44, relAl] for cloning of mutated M NaCl/10 mM EDTA/5% (vol/vol) glycerol/0.05% (wt/ genes. vol) bovine serum albumin at 37°C for0, 3, 6, 9, or 12 min with Construction of Mutant Genes. The synthetic gene encod- various concentrations of proteins and substrate. Reactions ing T4 endo V (11) was used to create mutants by replacement were stopped by addition of the SDS/piperidine solution to of restriction fragments. To facilitate this, four more unique a final concentration of 0.1% (wt/vol) and 0.1 M, respec- restriction sites-Spe I, Sac I, Nsp7524I, and Fsp I-were tively, and then the mixtures were boiled for 20 min to introduced into the gene by silent mutations of codons: convert abasic products (14-mer) to strand-cleaved forms Thr-7-Leu-8, ACTTTA -+ ACACTA; Glu-23-Leu-24, (6-mer and 7-mer). The cleaved products were separated GAATTG -> GAGCTC; Lys-33-His-34, AAGCAC -* AAA- from the residual substrate by DEAE-cellulose homochro- CAT; and Ala-123, GCA -* GCG. For construction of mutant matography (16), and the ratios were analyzed with a Fuji genes, a pUC19-derived cloning vector was used. The se- BA100 imaging plate. quences of the replaced fragments were confirmed by the Binding Assay by CD Spectroscopy. The mutant proteins chain-termination method (14), and the genes were trans- (R3Q, E23Q) were examined for their ability to bind the ferred to the expression vector pEnd V, which contained the substrate by CD spectroscopy, which measures the substrate E. coli trp promoter. oligonucleotide duplex (1.5 nmol) in the presence or absence Expression and Purification of T4 Endo V and the Mutant ofproteins (1.5 nmol) in 800 1l of32 mM Tris-HCl, pH 7.5/9.6 Proteins. E. coli cells containing the expression plasmid were mM EDTA/100 mM NaCl at 30°C. The spectra were taken in precultured in 75 ml of LB broth overnight at 30°C. The cells the range of 250-350 nm. Under these conditions, the CD were inoculated into 1 liter of fresh M9 medium, supple- band of the proteins was negligible. mented with 0.5% glucose and 0.2% Casamino acids, at a starting OD650 of 0.05-0.1, and were grown at 30°C to an RESULTS OD650 of0.5-0.9. Indoleacrylic acid was then added to a final Mutagenesis of the T4 Endo V Gene. To find the PD concentration of 20-40 jug/ml for induction of the trp pro- glycosylase active site of T4 endo V, extensive site-directed moter. The cells were cultured for another 2-3 hr and mutagenesis was performed on the following amino acids harvested. The cells were disrupted by sonication and the (Fig. 3): (i) basic and acidic residues around the aromatic lysates were centrifuged at 10,000 x g for 30 min at 4°C. The residue-rich sequence, Trp-Tyr-Lys-Tyr-Tyr (residues 128- clarified lysates (-30 ml) were applied to 20 ml of an 132), which is thought to participate in nucleotide binding; (ii) S-Sepharose fast flow column (Pharmacia). The proteins all arginine residues in the protein, to examine the effects on were eluted by a linear gradient of 0-0.75 M NaCl in 50 mM sodium phosphate buffer (pH 6.5). T4 endo V protein mutants eluted at 0.3-0.4 M NaCl. They were further purified by DNA affinity and phosphocellulose (P-11) column chro- matographies as described (15). The N-terminal 20-26 amino acid residues of the mutant proteins T2A, R3Q, R3K, R22Q, R22K, E23Q, E23D, R26Q, and R26K (Table 1) were sequenced by Edman degradation (model 477A; Applied Biosystems) to ensure that the N-ter- minal methionines were processed in E. coli and that residues mutated to glutamine remained intact during the purification procedure. (Endo V mutants are designated by the one-letter abbreviations for amino acids. The amino acid to the left of the residue number is the original amino acid, while the one to the right is the substituted amino acid.)

50

100 FIG. 3. Schematic drawing ofcrystal structure ofT4 endo V. The LGMMIVTFFYDKLEFLRKCROUAEaCLRGFNICDTT'VODISDI enzyme consists of three helices and loops. Shadow represents Haftx 2 region where basic side chains, such as Arg-3, Arg-22, Arg-26, Lys-80, Arg-117, Lys-121, Arg-125, etc. assemble at the surface of the protein. *, Mutant proteins D14N, R70Q, K80Q, R81Q, and R1OlQ did not accumulate in the cells in an amount large enough to FIG. 2. Amino acid sequence of T4 endo V. N-terminal methio- isolate; **, P25A protein was recovered from the insoluble fraction nine is processed in E. coli. Underlined sequences form a-helices. of cells. Downloaded by guest on October 1, 2021 9422 Biochemistry: Doi et al. Proc. Natl. Acad. Sci. USA 89 (1992) Table 1. PD glycosylase activities of T4 endo V mutant proteins ;6- CO mC. 0 vC~) cs (0 CD cm < cNi O~j V- r-,j Product/enzyme, Product/enzyme, cr mol per mol mol per mol > L11 LL Er Er: m: E 0 Mutant per min Mutant per min Wild type 3.1 (100) R3Q* ND E11Q 2.4 (76) R22Q* 0.2 (6) 43 H16Q 1.2 (39) R22K 1.5 (48) 29 Y21F 3.2 (103) R26Q* 0.03 (0.9) Sc18 E23Q* ND R32Q 2.9 (92) E23D* ND R40Q 2.6 (83) 6 K86Q 3.1 (100) R42Q 2.3 (74) D87N 2.5 (81) R68Q 2.0 (66) D87E 3.3 (106) R117Q 1.2 (40) '3 D119N 2.9 (95) R125Q 2.8 (89) E120Q 2.8 (90) K121Q 0.3 (9) FIG. 4. Analysis ofpurified mutant proteins. Mutant proteins and Y129F 1.1 (35) wild-type T4 endo V (WT) were purified by three column chromatog- Y132W 2.8 (89) raphies and analyzed by SDS/15% PAGE. Gel was stained with K134Q 2.5 (81) Coomassie brilliant blue. Size markers (x10-3) were prestained standards with calibrated molecular weights as follows: ovalbumin, In the steady-state assay, a substrate concentration of 200 nM and 43,000; carbonic anhydrase, 29,000; P-lactoglobulin, 18,400; lyso- an enzyme concentration of 0.6 nM were used as the standard zyme, 14,300; bovine trypsin inhibitor, 6200; insulin (A and B condition. ND, not detected. Activities relative to wild type are chains), 2300 and 3400. indicated in parentheses. *Enzyme concentration, 6.2 or 62 nM. Table 2 summarizes the kct and Km values of the different mutant proteins. The most serious defects were exhibited by specific substrate binding; and (iii) basic residues and Glu-23 the mutant proteins T2A, R3K, R22Q, R26Q, and R26K, in the basic amino acid cluster, because this structure seemed which had 7- to 200-fold decreases in k,,g and 9- to 200-fold to be unusual and interesting. increases in Km. Decreases by a factor of 1000 were observed Expression and Purification ofthe Mutant Proteins. Most of in the kct/Km values. These results indicate the importance the mutant proteins accumulated in the soluble fraction of of these residues both in substrate binding and in catalysis. and 5-10%o of cells, like the wild-type protein, represented The mutant proteins R117Q and K121Q also showed a 14- the total were protein. They purified by cation-exchange to 18-fold increase in Km, which reflected substrate-binding column chromatography, which gave >70% purity, and while the mutations resulted in small effects screened for PD glycosylase activity (see below). The mutant ability, relatively on In addition, the H16Q mutant protein exhibited a proteins that exhibited defective activities, as compared to k,.t. 2-fold increase in the Y129F protein showed a 4-fold were further by DNA affinity Km, the wild-type protein, purified in and the and exhibited and phosphocellulose column chromatographies. The proce- decrease kct, R68Q Q124A proteins dure yielded proteins of>95% purity (Fig. 4). The CD spectra kcat and Km values similar to those of the wild-type protein. Abilities ofthe Mutant Proteins and of the mutant proteins showed a typical a-helix Substrate-Binding E23Q purified The loss of PD the pattern in the region of200-250 nm (Fig. 5). The mean residue R3Q. complete glycosylase activity by substitution of for Glu-23 and Arg-3 allowed mea- ellipticities were roughly the same as that of the wild-type glutamine protein, 416,000 degree-cm2-dmol-1, indicating no substan- tial changes in the secondary structure. PD Glycosylase Activities of Mutant Proteins. Mutant pro- Wild type - Wild type teins were screened for PD glycosylase activity under steady- R26K --- E23D state conditions by using pools of the cation column chro- R22Q R30 matography fractions. The activity ofeach protein relative to the wild-type was calculated based on both activity and purity, as measured by high-performance liquid chromatog- raphy equipped with an Applied Biosystems Aquapore RP- 300 column. As shown in Table 1, the mutant proteins R3Q, E23Q, and E23D showed no PD glycosylase activity, even with a 10- or 100-fold higher enzyme concentration. The mutant proteins R22Q, R26Q, and K121Q showed relative activities that were <10% of the wild-type activity. The mutant proteins H16Q, R22K, R117Q, and Y129F exhibited 35-48% ofthe wild-type activity. Other mutant proteins showed activities similar to -20U000 1 I__ that ofthe wild type. All the mutations that yielded defective 200 250 200 250 T4 endo V proteins mapped to the basic amino acid cluster. Wavelength, nm Kinetic Analysis of Mutant Proteins. Although T4 endo V carries out two types ofreactions, the PD glycosylase activity FIG. 5. CD spectra of wild-type and mutant T4 endo V. CD could be analyzed as a Michaelis-Menten type ofreaction by spectra for wild-type and mutants (T2A, R3Q, R3K, R22Q, E23Q, E23D, R26Q, and R26K) were obtained with a J-60 automatic measuring the initial velocity and by controlling conditions so spectropolarimeter (Japan Spectroscopic, Tokyo). Measurements pro- that the concentration of apyrimidinic oligonucleotide were made at a protein concentration of0.15 mg/ml in 0.1 M NaCl/50 duced in the PD glycosylase reaction would be negligible mM sodium phosphate buffer, pH 6.5, at 20°C, using a cell with an compared to the thymidine dimer substrate concentration. optical path of 2 mm. Spectra for wild-type, R22Q, R26K, R3Q, and Also, the AP endonuclease reaction is much slower than the E23D proteins are shown. Observed ellipticity values are expressed PD glycosylase reaction. as mean residue ellipticity, [0], with the units degree-cm2-dmol1. Downloaded by guest on October 1, 2021 Biochemistry: Doi et al. Proc. Nat!. Acad. Sci. USA 89 (1992) 9423

Table 2. Kinetic parameters for PD glycosylase activities of T4 the substrate containing a thymidine dimer showed a typical endo V mutant proteins B-DNA-type spectrum in the absence of any protein (spec- kcat/Km, trum A), while, in the presence of the E23Q protein, the kcat, Km, M-1s-1 x Enzyme, Substrate, substrate displayed a quite different spectrum (spectrum B) min-' nM 103 nM nM from spectrum A, indicating the binding of the E23Q protein Wild type 4.3 48 1.5 x 103 0.31 20-100 to the substrate. On the other hand, the spectrum of the T2A 0.02 440 0.9 62 100-800 oligonucleotide without a thymidine dimer was not substan- R3K 0.6 >2000 <5.0 310 200-2000 tially affected by addition ofthe E23Q protein (spectrum E in H16Q 4.2 110 6.3 x 102 0.31 50-1600 Fig. 6III). The difference between spectra D and E (spectrum R22Q 0.32 310 1.7 x 101 31 100-800 F in Fig. 6IV) could be due to nonspecific binding ofthe E23Q R22K 3.2 330 1.6 x 102 0.62 75-1600 protein to DNA. R26Q 0.06 770 1.3 31 100-1200 The same experiments were performed with the R3Q R26K 0.2 1100 2.4 62 200-1200 mutant protein (Fig. 6V). Addition ofthe R3Q protein did not R68Q 4.2 40 1.8 x 103 0.31 20-100 affect the spectrum of the substrate at all (spectrum H), R117Q 2.2 670 5.5 x 101 0.62 30-800 indicating a lack of specific binding by the R3Q protein to the E120Q 2.7 28 1.6 x 103 0.31 20-100 substrate. Fig. 6V1 shows that the presence of T4 endo V K121Q 11.0 890 2.0 x 102 0.62 75-2000 wild-type protein did not affect the CD spectrum of the Q124A 3.8 31 2.0 x 103 0.62 20-400 oligonucleotide lacking a pyrimidine dimer, suggesting that Y129F 1.1 43 4.3 x 102 0.62 20-800 binding does not occur under the conditions used. Five different points were taken in the indicated range of substrate concentration. Under these conditions, the apyrimidinic product DISCUSSION concentration was <10% of the original thymidine dimer substrate concentration during the first 12 min of incubation. T4 endo V, a pyrimidine dimer-specific excision repair en- zyme, is endowed with both PD glycosylase and AP endo- surement of the CD spectra of the oligonucleotide substrate nuclease activities. In addition, this enzyme is capable of in the absence or presence of equimolar amounts of mutant scanning or sliding along DNA in a processive manner for proteins, since the substrate remained undigested during the effective recognition of a pyrimidine photodimer in DNA (4). measurements. Therefore, the differences in their spectra are To dissect the complex reaction mechanisms of T4 endo V, attributable to the conformational changes in oligonucleotide we have focused on PD glycosylase activity and carried out that arise from its binding to the protein. As shown in Fig. 6I, a systematic site-directed mutagenesis aimed at identification

0 x 0; E230 w ( (E3 A IV P, E-D VI(W K1.-T4T4dbmVwwubVW±L(WV) L;K-J

1 D1IL1

F L 0

-1iS -1

d'oc~c 1-.GCACG3 d'CGcAC= G CICACG' d CGTGC CGTC d CG0GC CGTGC5 250 )E;300 350 250 Soo 350 250 300 350 250 30 350 Wavelength, nm FIG. 6. Substrate binding by E23Q and R3Q mutant proteins. CD spectra of the oligonucleotide complexes indicated were measured in the absence (-) or presence (+) of the protein. Each spectrum corresponds to the following: A, oligonucleotide substrate containing a thymidine dimer; B, thymidine dimer substrate with the E23Q protein; C, difference spectrum of A and B; D, oligonucleotide duplex with no thymidine dimer; E, same oligonucleotide duplex as spectrum D in the presence of E23Q protein; F, difference spectrum of D and E; G, same as spectrum A; H, same as spectrum A in the presence of R3Q; I, difference spectrum of H and G; J, same as spectrum D; K, same as spectrum D in the presence of wild-type T4 endo V protein; L, difference spectrum of J and K. Downloaded by guest on October 1, 2021 9424 Biochemistry: Doi et al. Proc. Natl. Acad. Sci. USA 89 (1992)

of an active site in the enzyme (Fig. 7). Based on knowledge change around Glu-23, resulting in the decrease in k" t. As for from the x-ray crystal structure of the enzyme and the the effect on Ki, we favor the interpretation that a side chain biochemical evidence obtained so far, our investigation con- of Arg-26 is directly involved in substrate binding, since it is centrated on residues near the Trp-Tyr-Lys-Tyr-Tyr (resi- located close to Arg-3 and Glu-23. Other mutant proteins- dues 128-132) sequence and in or near the basic amino acid R22K, R22Q, R17Q, and K121Q-exhibited a 7- to 18-fold cluster. Furthermore, all arginine residues were systemati- increase in Km, indicating the involvement of those residues cally substituted to examine their effects on the substrate- in substrate binding. The difference between the kct values binding ability of the enzyme. ofthe R22K and R22Q proteins points out the requirement for The PD glycosylase activities of 29 mutant proteins were a positive charge at the position of Arg-22. This could imply that a certain electrostatic balance in the region surrounding analyzed by using a 14-mer oligonucleotide substrate that Glu-23 is important for catalysis. The side chains of Arg-117 contained a single thymidine dimer. In the steady-state assay and Arg-3 hydrogen bond to the same water molecule. The (Table 1), the mutant proteins R3Q, E23Q, and E23D did not low affinity of the R117Q protein for the substrate might be exhibit any activities. Furthermore, the E23Q protein re- due to a direct effect on substrate binding or to an effect tained a substrate-binding ability in a specific manner (Fig. 6), through Arg-3. The side chains of Arg-22, Arg-26, Arg-117, whereas R3Q protein did not. The PD glycosylase activity of and Lys-121, which displayed defects in substrate binding, the R3K protein was also found to be seriously impaired. The are all located in the close vicinity of the side chain of Arg-3. defects caused by this mutation seemed to be more serious This also indicates the importance of this region in substrate for substrate binding than for catalysis. This finding is binding. consistent with the complete loss of substrate-binding ability Here, we show that Glu-23 is an indispensable residue for in the R3Q mutant protein. X-ray crystallography has shown catalysis of the PD glycosylase reaction and that Arg-3 is that the E23Q and R3Q proteins possess a three-dimensional essential for substrate binding. In addition, Arg-22, Arg-26, structure similar to that of wild type and that the changes in Arg-117, and Lys-121 participate in substrate binding. It is protein structure due to the mutation are confined to the side evident that the basic amino acid cluster of T4 endo V is a chain ofthe altered amino acid. We conclude that Glu-23 and fundamental structure for PD glycosylase activity. In the Arg-3 are essential residues for catalysis of the PD glycosy- crystal structure of T4 endo V, a groove-like depression is lase reaction and binding of the pyrimidine dimer substrate, observed near the Trp-Tyr-Lys-Tyr-Tyr (residues 128-132) respectively. Considering that neither the E23Q nor the E23D sequence and the central region of the basic amino acid proteins showed PD glycosylase activity, a strict tertiary cluster, into which the DNA could be accommodated. The structure around the carboxyl group of Glu-23 must be crystal structure of T4 endo V and the pyrimidine dimer required for the reaction. Assuming general acid catalysis for substrate complex should clarify how the protein binds to the the PD glycosylase reaction (2), it is probable that the substrate. carboxyl group of Glu-23 functions to donate a proton. We thank Dr. K. Katayanagi and Ms. Ariyoshi for production of A serious defect that affected both the kct and Km values the molecular graphics representation of T4 endo V. This research was observed in the T2A mutant protein and was probably was supported in part by a grant-in-aid from the Ministry of Educa- due to perturbation ofthe structure around Arg-3 and Glu-23. tion, Science and Culture of Japan. A similar type of defect was observed in R26Q and R26K mutant proteins. In the crystal structure, the side-chain 1. Friedberg, E. C. (1985) DNA Repair (Freeman, New York). of forms a with the 2. Dodson, M. L. & Lloyd, R. S. (1989) Mutat. Res. 218, 49-65. 8-guanidino group Arg-26 hydrogen bond 3. Valerie, K., Henderson, E. E. & deRiel, J. K. (1984) Nucleic backbone carbonyl group of Arg-22 in the helix. Therefore, Acids Res. 12, 8085-8096. lack of the hydrogen bond might cause a minute structural 4. Gruskin, E. A. & Lloyd, R. S. (1988) J. Biol. Chem. 263, 12728-12737. 5. Belmoaras, T., Toulme, J.-J. & Helene, C. (1981) Proc. Nat!. Acad. Sci. USA 78, 926-930. 6. Stump, D. G. & Lloyd, R. S. (1988) Biochemistry 27, 1839- 1843. 7. Ishida, M., Kanamori, Y., Hori, N., Inaoka, T. & Ohtsuka, E. (1990) Biochemistry 29, 3817-3821. 8. Dowd, D. R. & Lloyd, R. S. (1989) Biochemistry 28, 8699- 8705. 9. Dowd, D. R. & Lloyd, R. S. (1989) J. Mol. Biol. 208, 701-707. 10. Dowd, D. R. & Lloyd, R. S. (1990) J. Biol. Chem. 265, 3424- 3431. 11. Inaoka, T., Ishida, M. & Ohtsuka, E. (1989) J. Biol. Chem. 264, 2609-2614. 12. Morikawa, K., Tsujimoto, M., Ikehara, M., Inaoka, T. & Ohtsuka, E. (1988) J. Mol. Biol. 202, 683-684. 13. Morikawa, K., Matsumoto, O., Tsujimoto, M., Katayanagi, K., Ariyoshi, M., Doi, T., Ikehara, M., Inaoka, T. & Ohtsuka, E. (1992) Science 256, 523-526. 14. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Nat!. FIG. 7. Molecular graphic representation of the active site of T4 Acad. Sci. USA 74, 5463-5467. endo V, using the program FRODO on an Evans and Sutherland PS 390 15. Nakabeppu, Y., Yamashita, K. & Sekiguchi, M. (1982) J. Biol. graphics system. Every 10th Ca atoms is numbered and the N Chem. 257, 2556-2562. terminus is denoted by N. Side chains shown are Arg-3, Arg-22, 16. Jay, E., Bambara, R., Padmanabhan, R. & Wu, R. (1974) Glu-23, and Arg-26. Nucleic Acids Res. 1, 331-353. Downloaded by guest on October 1, 2021