Physical Association of Pyrimidine Dimer DNA Glycosylase and Apurinic/Apyrimidinic DNAEndonuclease Essential for Repair of Ultraviolet-Damaged DNAAuthor(s): Yusaku Nakabeppu and Mutsuo SekiguchiSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 78, No. 5, [Part 2: Biological Sciences] (May, 1981), pp. 2742-2746Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/10694 .

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Proc. Natl. Acad. Sci. USA Vol. 78, No. 5, pp. 2742-2746, May 1981 Biochemistry

Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA

(bacteriophage/T4 endonuclease V/multifunctional enzyme)

YUSAKU NAKABEPPU AND MUTSUO SEKIGUCHI*

Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, Japan

Communicated by Seymour S. Cohen, January 22, 1981

ABSTRACT T4 endonuclease V [endodeoxyribonuclease (py- rimidine dimer), EC 3.1.25.1], which is involved in repair of UV- damaged DNA, has been purified to apparent physical homo- geneity. Incubation of UV-irradiated poly(dA)poly(dT) with the purified enzyme preparations resulted in production of alkali-la- bile apyrimidinic sites, followed by formation of nicks in the poly- mer. The activity to produce alkali-labile sites was optimal in a relatively broad pH range (pH 6.0-8.5), whereas the activity to form nicks had a narrow optimum near pH 6.5. By performing a limited reaction with T4 endonuclease V at pH 8.5, irradiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks. The inter- mediate was used as substrate for the assay of apurinic/apyr- imidinic DNA endonuclease activity [endodeoxyribonuclease (apurinic or apyrimidinic, EC 2.1.25.2]. The two activities, a py- rimidine dimer DNA glycosylase and an apurinic/apyrimidinic DNA endonuclease, were copurified and found in enzyme prep- arations that contained only a 16,000-dalton polypeptide. An en- zyme fraction from cells infected with bacteriophage T4v,, a mu- tant that is sensitive to UV radiation, was defective in both glycosylase and endonuclease activities. Moreover, occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities, and suppression of the mutation rendered both activities partially active. These results strongly suggested that a DNA glycosylase specific for pyrimidine dimers and an apurinic/apyrimidinic DNA endonuclease reside in a single poly- peptide chain coded by the denV gene of bacteriophage T4. Be- cause the two activities exhibited different thermosensitivity, it was further suggested that conformation of the active sites for these activities may be different.

Strand scission of DNA in the vicinity of pyrimidine dimers appears to be the necessary first event for excision repair of UV- damaged DNA (1-3). Evidence has been presented that T4 en- donuclease V [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] is responsible for this step in bacteriophage T4-in- fected Escherichia coli (4, 5). The enzyme activity is induced after infection of cells with wild-type phage T4 but not with the T4v1 mutant, which is sensitive to UV light (6). Studies with temperature-sensitive v mutants have revealed that the enzyme is indeed coded by the denV gene (v gene) of phage T4 (7).

T4 endonuclease V is specifically active on UV-irradiated DNA and does not act on DNA that is damaged by other agents, such as 4-nitroquinoline-N-oxide and methyl methanesulfonate (8, 9). It seems that the enzyme recognizes a UV-induced py- rimidine dimer in DNA and induces a single-strand break near the dimer (4, 5, 10, 11). UV endonuclease from Micrococcus luteus has been shown to possess similar properties (12, 13).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. ? 1734 solely to indicate this fact.

Because of their high substrate specificity, the enzymes have been of extensive use for both quantification of pyrimidine di- mers in DNA and analysis of cellular repair mechanisms (14, 15).

Grossman et al. (16) and Haseltine et al. (17) recently have demonstrated that UV endonuclease of M. luteus possesses DNA glycosylase activity which cleaves the glycosylic bond be- tween the 5'-pyrimidine of a dimer and the corresponding deox- yribose. They have indicated that T4 endonuclease V also has such an activity (17), and this has been confirmed by others (18-21).

Because the glycosylic cleavage itself does not cause inter- ruption in the phosphodiester backbone, an additional catalytic step is required to produce strand scission. Indeed, apurinic/ apyrimidinic (AP) DNA endonuclease [endodeoxyribonuclease (apurine or apyrimidine), EC 2.1.25.2] activity, which can act on the AP sites generated in DNA by the glycosylase, was found in enzyme preparations from both M. luteus and phage T4-in- fected E. coli cells (16-21). Thus, a question arises whether the glycosylase and the AP DNA endonuclease are fortuitously as- sociated or integral parts of a single repair enzyme.

To resolve this problem, it is necessary to purify the enzyme to physical homogeneity. In this paper we report that T4 en- donuclease V was purified to the state of apparent homogeneity. With such purified enzyme preparations we obtained evidence to show that the two distinct catalytic activities are carried by a single polypeptide chain. Genetic evidence that supports this view is also presented.

MATERIALS AND METHODS

Bacteria and Bacteriophages. E. coli strains 1100 (Endl-Su+), B(Su-), and CR63(Su+) were used in these experiments. Bac- teriophage T4D and T4vj were provided by W. Harm (6). Bac- teriophage T4uvs-5, a T4 mutant having an amber mutation in the denV gene (22), was obtained from L. van Minderhout.

Chemicals and Enzyme. E. coli DNA polymerase I and poly(dA)poly(dT) were purchased from Bethesda Research Laboratories (Rockville, MD) and Sigma, respectively. [methyl- 3H]dTTP was obtained from New England Nuclear.

Preparation of DNA and Polynucleotide. 32P-Labeled phage T4 DNA was prepared as described (4). [3H]Thymine-labeled poly(dA)poly(dT) was prepared as follows. The reaction mix- ture (2.2 ml) contained 760 nmol (nucleotide equivalent) of poly(dA)poly(dT), 140 ,tmol of potassium phosphate (pH 7.5), 2.1 ,tmol of 2-mercaptoethanol, 14 ,tmol of MgCl2, 1.6 ,tmol of [3H]dTTP (50 ,tCi/,tmol; 1 Ci = 3.7 x 1010 becquerels), 1.6

Abbreviations: AP, apurinic/apyrimidinic; PEME, phosphate/ethyl- ene glycol/2-mercaptoethanol/EDTA buffer. * To whom reprint requests should be addressed.

2742

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Biochemistry: Nakabeppu and Sekiguchi Proc. Natl. Acad. Sci. USA 78 (1981) 2743

17 6 V

*... .. 1. ;'. '1'

13

1 2 3 4

FIG. 1. NaDodSO4/polyacrylamide gel electrophoresis of crude or purified enzyme fractions from T4D-infected and T4vj-infected cells. Samples were run at 20 mA and stained with Comassie-brilliant blue. Migration was from top to bottom. Tracks: 1, fraction III' from phage T4D-infected E. coli 1100; 2, fraction III' from phage T4v1-infected E. coli 1100; 3, fraction VI from phage T4D-infected E. coli 1100; 4, chy- motrypsinogen (25,000 daltons), myoglobin (17,000 daltons), and cy- tochrome c (13,000 daltons), shown in kilodaltons.

,mol of dATP, and 24 units of E. coli DNA polymerase I. The mixture was incubated at 37?C for about 10 hr and then shaken with phenol. The aqueous layer containing the polymer was dialyzed against six 1-liter changes of 1 mM EDTA/0. 1 M NaCl/ 10 mM Tris HCl, pH 7.5, at 0?C for 2 days. The polymer at a concentration of 180 nmol/ml in the buffer was irradiated with a 15-W germicidal lamp (Toshiba) at room temperature. Irra- diation was usually for 10 min at a distance of 10 cm from the lamp (approximate dose, 4200 J/m2).

To prepare polynucleotide that has AP sites at the 5' pyrim- idine of dimers, [3H]thymine-labeled, UV-irradiated poly(dA)-poly(dT) (200 nmol) was treated with 6.5 units of T4 endonuclease V (fraction VI) in 1. 8 ml of 9.6 mM EDTA/32 m M Tris*HCl, pH 8.5, at 37?C for 10 min. The treated polymer was shaken with phenol and dialyzed against four 0.5-liter changes of 1 mM EDTA/0. 1 M NaCV10 mM Tris HCI, pH 7.5, at 0?C overnight.

Assay of Enzyme Activities. T4 endonuclease V activity was determined by measuring degradation of 32P-labeled, UV-ir- radiated phage T4 DNA in the presence of an extract of mutant T4vj-infected E. coli 1100 (11). This assay was used throughout purification of the enzyme.

The assay of pyrimidine dimer DNA glycosylase activity de- pends on formation of alkali-labile sites in irradiated synthetic polymer. The reaction mixture (100 ,ul) contained 0.6 nmol of [3H]thymine-labeled, UV-irradiated poly(dA)poly(dT) (3.6 x 104 dpm/nmol), 3.2 Amol of Tris HCl (pH 7.5), 0.96 Amol of EDTA, and an enzyme. Incubation was at 37?C for 20 min un- less otherwise indicated. At the end of incubation, 20 ,tl of 1 M NaOH was added (final pH, 13) and the mixture was further incubated at 37?C for 60 min. After alkali treatment, the mixture was chilled and precipitated with the addition of 50 Al of carrier DNA (1 mg/ml) and 30 ,ul of 50% (wt/vol) trichloroacetic acid. After standing for 30 min at 0?C, the sample was centrifuged and the supernatant solution was taken as the acid-soluble frac- tion. The radioactivity was determined in a liquid scintillation counter.

For the assay of AP D NA endonuclease, irradiated poly(dA) polyv(dT) that had been treated with T4 endonuclease at pH 8.5 was used as substrate. The conditions and the pro-

cedures were essentially the same as those for the assay of gly- cosylase, except that alkali treatment was omitted.

Purification of T4 Endonuclease V. T4 endonuclease V was purified from phage T4-infected E. coli through phase partition and CM-Sephadex chromatography as described (11). The CM- Sephadex fraction (fraction III) was concentrated and applied to a column of Bio-Gel P10 (1.5 x 100 cm) equilibrated with PEME (0.01 M potassium phosphate, pH 6.5/10% (voVvol) ethylene glycol/0.01 M 2-mercaptoethanol/2 mM EDTA) con- taining 0.5 M KCI. The column was eluted with the same buffer (flow rate, 5 ml/hr), and the active fractions were pooled (frac- tion IV).

Fraction IV (7.25 ml) was diluted with PEME to 0.2 M KC1 and applied to a column of phosphocellulose (0. 9 x 15 cm) equil- ibrated with PEME containing 0.2 KCI. The column was washed with 20 ml of the same buffer, followed by a linear gra- dient of 200 ml of the buffer with limits of 0.2 and 0.6 M KC1. The flow rate was 6 mVhr, and 3-ml fractions were collected. Fractions (12 ml) with specific activity greater than 1000 units/ mg of protein were pooled (fraction V).

Fraction V (11 ml) was diluted with PEME to 0.2 M KC1 and applied to a column (0.5 x 7 cm) of UV-irradiated DNA-cel- lulose (11) preequilibrated with PEME containing 0.2 M KCI. After washing the column with 10 ml of the same buffer, 80 ml of PEME containing a linear gradient of 0.2-1.0 M KC1 was applied. The enzyme activity appeared after passage of 18 ml

100 .

80

s 60

40

20

0 1.0 2.0

Enzyme, units/ml

FIG. 2. Formation of alkali-labile sites and nicks by T4 endonu- clease V. The reaction mixture contained 0.6 nmol of [3H]thymine-la- beled, UV-irradiated (4200 J/m2) poly(dA)*poly(dT) (3.6 x 104 dpm/ nmol), 3.2 ,umol of Tris HCl (pH 7.5), 0.96 ,pmol of EDTA, and various amounts of T4 endonuclease V (fraction VI) in 0.1 ml. After incubation at 370C for 20 mi, the samples were chilled and divided into two por- tions; one was directly acidified, and the other was acidified after treat- ment with alkali. *, Without alkali treatment; o, after alkali treatment.

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2744 Biochemistry: Nakabeppu and Sekiguchi Proc. Natl. Acad. Sci. USA 78 (1981)

50

40

, 30

20

10

0 5.0 6.0 7.0 8.0 9.0

pH

FIG. 3. Effect of pH on activities of T4 endonuclease V. Irradiated poly(dA).poly(dT) was treated with purified T4 endonuclease V (frac- tion VI; 0.16 unit/ml) in 32 mM Tris maleate/NaOH/9.6 mM EDTA at various pHs for 20 min at 37?C. Other conditions and procedures were as described in Fig. 2. e, Without alkali treatment; o, after alkali treatment.

of the gradient solution. The active fractions (6 ml) were pooled (fraction VI).

For comparison of samples from T4D-infected and T4v1-in- fected cells, crude enzyme fractions were prepared by a mod- ification of the procedure of Yasuda and Sekiguchi (11). A step- wise elution from a CM-Sephadex C-25 column (1 x 15 cm) was used rather than a linear gradient. After washing the column with 50 ml of PEME containing 0.2 M KCI, the column was eluted with 50 ml of PEME containing 0.4 M KCI, and 5-ml fractions were collected. The first 5-ml fraction was used as frac- tion III'.

Other Methods. NaDodSO4/15% polyacrylamide gel elec- trophoresis was essentially as described (23). Protein was de- termined by the method of Lowry et al. (24); in the case of highly purified fractions, the fluorescence method (25) was used.

RESULTS

Enzyme Activities Associated with Purified T4 Endonucle- ase V. T4 endonuclease V was purified to physical homogeneity. Only a single major band was detected when the purified en- zyme preparation (fraction VI) was analyzed by NaDodSO4/ polyacrylamide gel electrophoresis (Fig. 1). The corresponding band was found in a crude enzyme fraction (fraction III') from phage T4D-infected cells but not in a fraction from phage T4v1- infected cells. From the electrophoretic mobility, the molecular size of the polypeptide was estimated to be approximately 16,000 daltons, which is close to the value obtained on the basis of elution from a Sephadex C-5O column.

The ability of homogeneous preparations of T4 endonuclease V to produce both alkali-labile sites and nicks was demonstrated

40 - * 80

~30 z ~60

1.0-20 Z

20 - 40

0 ~~~~~~~~~~0.5-10 10 20- 10

-~~~~~~~~~~~~~

0 0 1010 1 5 10 15 20 25 30 35 40

Fraction

FIG. 4. Chromatography on irradiated DNA-cellulose of T4 en- donuclease V. o, T4 endonuclease V activity as determined with 32p- labeled, UV-irradiated phage T4 DNA substrate; e, pyrimidine dimer DNA glycosylase activity as measured with 3H-labeled, UV-irradiated poly(dA)*poly(dT) substrate; Ew, AP DNA endonuclease activity as de- termined using 3H-labeled, UV-irradiated poly(dA)*poly(dT) that had been subjected to a limited reaction with T4 endonuclease V at pH 8.5; *, amount of protein measured by fluorometric assay; x, concentration of KCI.

with poly(dA).poly(dT) irradiated with UV. Alkali-labile sites were first formed, which were converted to nicks by the sec- ondary reaction (Fig. 2). No alkali-labile sites or nicks were pro- duced in nonirradiated polymer incubated with the enzyme preparation, and extents of the reactions increased with in- creasing doses of UV (up to 4000 J/m2) applied to the polymer (data not shown).

Isolation of an Intermediate. Fig. 3 shows the effect of pH on the two activities associated with T4 endonuclease V. The activity to produce alkali-labile sites was active in a relatively broad pH range, from pH 6.0 to 8.5. In contrast, the activity to form nicks had a narrow pH optimum near pH 6.5 and was almost inactive at pH 8.0 or higher. Thus, when a limited re- action with T4 endonuclease V was performed at pH 8.5, ir- radiated polymer was converted to an intermediate form that carried a large number of alkali-labile sites but only a few nicks.

The intermediate thus formed seemed to possess apyrimi- dinic sites together with pyrimidine dimers, whose 3'-pyrimi- dine is attached to the polymer, as proposed by Haseltine et al. (17) (see Fig. 8). This notion is supported by the following lines of evidence, details of which will be published elsewhere. (i) Reduction with sodium borohydride decreased greatly the sen- sitivity of the intermediate to alkali. (ii) Free thymine was re- leased from the polymer when it was subjected to direct pho- toreversal with 254-nm light.

Copurification of Activities. The foregoing experiments in- dicated that purified preparations of T4 endonuclease V possess a DNA glycosylase activity specific for pyrimidine dimer and an AP DNA endonuclease activity. By using the intermediate as substrate, it is possible to assay the AP DNA endonuclease activity independent of the glycosylase.

Fig. 4 shows elution profiles of activities from a UV-irradiated DNA-cellulose column. Peaks of the glycosylase and the AP DNA endonuclease activities coincided well with a peak of pro- tein and also with a peak of T4 endonuclease V, which was de- termined by a conventional method. It was shown, moreover, that ratio of the AP DNA endonuclease activity to the glyco- sylase activity was constant through the last two steps of purification.

Heat Inactivation of Activities. A highly purified preparation of T4 endonuclease V was kept at 42?C for various times, and

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Biochemistry: Nakabeppu and Sekiguchi Proc. Natl. Acad. Sci. USA 78 (1981) 2745

100

0 p

2t50 tX

0 10 20 Preincubation time, min

FIG. 5. Heat inactivation of T4 endonuclease V. A purified prep- aration of T4 endonuclease V (fraction VI; 0.32 unit/ml) was incubated in 10 mM Tris HCI, pH 7.5/1 mM 2-mercaptoethanol at 42?C. At var- ious times, samples were withdrawn and activities were determined at 37?C. o, Pyrimidine dimer DNA glycosylase; *, AP DNA endonuclease.

both glycosylase and AP DNA endonuclease activities were as- sayed at 37?C. As shown in Fig. 5, the AP DNA endonuclease activity was destroyed very rapidly, whereas the glycosylase activity was lost at a linear rate of 4% per min. Thus, the AP DNA endonuclease activity is more heat labile than the gly- cosylase activity.

Alteration of Enzyme Activities with T4 Mutations. We pre- pared crude enzyme preparations (fraction III') from T4D-in- fected cells and from T4vl-infected cells and compared their enzyme activities. Both glycosylase and AP DNA endonuclease activities were completely absent from the enzyme fraction from phage T4vl-infected cells (Fig. 6).

A close examination of gel electrophoretic patterns of the two enzyme preparations (see Fig. 1) revealed that the v1 sample was devoid of a 23,000-dalton polypeptide in addition to a 16,000-dalton polypeptide (T4 endonuclease V). However, it is unlikely that one of the activities, glycosylase or AP DNA endonuclease, resides in a 23,000-dalton polypeptide because the corresponding band was not found in purified preparations.

The above data suggested that the T4v, mutant might have two independent mutations or a small deletion that covers the denV and an adjacent gene(s). To make sure that the two activ- ities are controlled by one gene, we performed an additional experiment with a phage T4 mutant having a single mutation in the denV gene. Phage T4uvs-5, which possesses an amber mutation in the denV gene (22), exhibited normal UV sensitivity when plated on E. coli strain CR63(Su') but was as sensitive as phage T4v, on strain B(Su-).

Crude extracts were prepared from uninfected cells and from cells infected with phages T4D, T4v, or T4uvs-5, and enzyme activities were determined (Fig. 7). Levels of both glycosylase and AP DNA endonuclease activities in T4uvs-5-infected E . coli B were essentially the same as those of uninfected cells or T4v;1-

100 100 A B

Z 75 75 7

e- 50 50

D2 5_ 25>

0 0

0 5 10 0 5 10 Protein, yg/ml

FIG. 6. Enzyme activities of T4D-infected and T4vj-infected cells. Enzyme fractions (fraction III') from T4D-infected and T4v1-infected E coli 1100 were used for assays of pyrimidine dimer DNA glycosylase (A) and of AP DNA endonuclease (B). o, phage T4D-infected cells; *, T4vj-infected cells.

infected cells. When the amber mutation was suppressed by the supD mutation in strain CR63, significant increases in the two activities were observed. Thus, a single mutation and its suppression affected the two activities in an identical manner.

DISCUSSION

A two-step mechanism for the incision of DNA containing py- rimidine dimers has been proposed by Grossman et al. (16, 17) and by others (18-21). Based on several lines of evidence, the following reaction scheme was suggested: a cleavage of the N-

A C

60 60

40 40 c, cc 0~~~~~~~~~~~~~~~~~~~~~~~~~c

(o 20 20 U

a<> B D

O 60 60

z 40 40 C:

20 20

0 4A-- 0 0 0.1 0.2 0.3 0 0.1 0.2 0.3

Protein, mg/ml

FIG. 7. Enzyme activities in E. coli B(Su-) and CR63(Su+) cells infected with various phage T4 strains. Extracts were prepared from normal and phage-infected cells and activities of pyrimidine dimer DNA glycosylase and APDNA endonuclease were determined. (A) Glycosylase in strain B. (B) Glycosylase in strain CR63. (C) APDNA endonuclease in strain B. (D) APDNA endonuclease in strain CR63. o, T4D-infected cells; *, T4uvs-5-infected cells; A, T4v1-infected cells; *, noninfected cells.

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2746 Biochemistry: Nakabeppu and Sekiguchi Proc. Natl. Acad. Sci. USA 78 (1981)

52P-P 2NS P" "-~ 3'

Pyrimidine dimer DNA glycosylase

101, ~~~~T4 /?) (?) C' (H) /H)endonuclease V

V AP DNA endonuclease

5t P PdOH p 3,

FIG. 8. A model forthe action of T4 endonuclease V. The model was drawn on the basis of the data presented in this paper and by others (17-21).

glycosyl bond between the 5'-pyrimidine of a dimer and the corresponding sugar first takes place, and then a phosphodiester bond on the 3'-side of the AP site is cleaved (Fig. 8). The data presented in this paper also support this view.

Two enzyme activities, a pyrimidine dimer DNA glycosylase and an AP DNA endonuclease, have been found in highly pu- rified preparations of both M. luteus and phage T4 UV endo- nuclease (17-21). However, it has not been possible to ascertain whether they are fortuitously associated with enzyme prepa- rations or are integral parts of a single enzyme because homo- geneous prepar,ations of the enzymes have not been available. Because there was the selective loss of the AP DNA endonu- clease activity of the M. luteus enzyme relative to that of the pyrimidine dimer DNA glycosylase on purification, Haseltine et al. (17) suggested that the two activities may be separable. On the other hand, on the basis of the finding that the T4 en- zyme lost both UV and AP DNA endonuclease activities at the same rate during storage, Demple and Linn (20) have suggested that the AP DNA endonuclease and the glycosylase are com- panion activities in repair complexes or even single polypeptide chains.

In experiments designed to determine which of the activities found in T4 endonuclease V preparations is coded by the denV gene, Seawell et al. (21) have found that the glycosylase was more heat labile in extracts of E. coli infected with thermosen- sitive v mutants than in extracts of cells infected with wild-type phage T4. In contrast, AP DNA endonuclease activity was no more heat labile in extracts of the former than of the latter. The simplest interpretation of these data is that the denV gene codes for the glycosylase but not for the AP DNA endonuclease. How- ever, the possibility has remained that although both activities reside in the same protein, the ts mutations used are at positions that affect only the glycosylase but not the AP DNA endonu- clease. In such a case, it is possible that the AP DNA endo- nuclease is coded also by the denV gene.

We have shown in the present study that homogeneous prep- arations of T4 endonuclease V possess both pyrimidine dimer DNA glycosylase and AP DNA endonuclease activities. Ratio of the two activities did not change in the last two steps of pu- rification. Moreover, introduction of an amber mutation in the

denV gene caused a simultaneous loss of the two activities, and suppression of the mutation rendered both activities partially active. These results strongly suggest that the glycosylase and the AP DNA endonuclease must share a common polypeptide chain.

There is an obvious advantage of a multifunctional enzyme in a series of connected reactions in which the product formed by one reaction becomes available to the next at a high con- centration. In view of the remarkable similarity in the prop- erties of T4 endonuclease V and M. luteus UV endonuclease, it is likely that the M. luteus enzyme also carries a pyrimidine dimer DNA glycosylase and an AP DNA endonuclease in a sin- gle molecule, though definite evidence has not been available.

The AP DNA endonuclease activity associated with T4 en- donuclease V is more thermolabile than is the glycosylase ac- tivity associated with the same enzyme. The simplest expla- nation for this phenomenon is that there are two distinct active sites or domains in a single enzyme, each of which corresponds to one of the activities. However, it might be difficult to assume two independent domains for a small protein molecule, such as T4 endonuclease V (16,000 daltons). It may be supposed that the two activities are handled by one active center but differ in degree of dependence on conformation of the center.

This work was supported by Scientific Research and Cancer Research grants from the Ministry of Education, Science and Culture of Japan.

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