The DNA polymerase β reaction with ultraviolet-irradiated DNA incised by correndonuclease

246

Biochimica et Biophysica Acta, 609 (1980) 246--256

© Elsevier/North-Holland Biomedical Press

BBA 99718 THE DNA POLYMERASE/] REACTION WITH ULTRAVIOLET-IRRADIATED DNA INCISED BY CORRENDONUCLEASE

RADOSLAWA NOWAK a, ZOFIA ZAREBSKA a and BARBARA ZMUDZKA b a Institute of Oncology, Wawelska, 15, 02-034 Warsaw and b Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Rakowieka 36, 02-356 Warsaw (Poland)

(Received January 23rd, 1980) Key words: DNA polymerase ~; Correndonuclease; Radiation damage; DNA repair mechanism; (Calf thymus)

Summary Covalently closed circular Col E1 DNA was ultraviolet-irradiated with a dose of 60 J / m 2, thus introducing about 3.2 pyrimidine dimers per DNA molecule. Treatment of irradiated Col E1 DNA with M i c r o c o c c u s l u t e u s correndonuclease resulted, in the vicinity of pyrimidine dimers, in an average of 3.3 incisions per DNA molecule, and converted DNA to the open circular form. Incised Col E1 DNA stimulated no reaction with calf t h y m u s DNA polymerase ~ but was recognized as a template by DNA polymerase/3. The latter enzyme incorporated about 1.6 molecules of dTMP (corresponding to 6 molecules of dNMP) per one correndonuclease incision. The length of the DNA polymerase fi product was comparable to the anticipated length of the DNA region within which the hydrogen bonds were disrupted owing to dimer formation. The enzyme required Mg2÷ and four dNTPs for reaction and was resistant to N-ethylmaleimide or p-mercuribenzoate. The average numbers of deoxynucleotides incorporated per one DNAase I incision or per one nonspecific break, measured in control samples, were equal, amounting to 0.3 dTMP molecule. This value corresponded to 1.2 dNMP molecule; in our opinion, this reflects contaminating nuclease activity of the system used. The present results testify to the ability of DNA polymerase /~ to repair synthesis by the 'patch and cut' mechanism.

247 Introduction Results of various experimental approaches point to DNA polymerase a (EC 2.7.7.7) as the enzyme responsible for semiconservative DNA replication in mammalian cell nuclei [1--6]. However some experimental data suggest that DNA polymerase a participates also in both postreplication and excision repair, (viz.) it replicates dimer-containing template [ 7,8] and fills small gaps caused in depurinated DNA by repair endo- and exonuclease [9]. Furthermore, the properties of the enzyme responsible for ultraviolet-induced repair replication in WR 38 cell nuclei and in HeLa cells resemble DNA polymerase a characteristics [10,11]. Mitochondrial DNA polymerase, which may be identical with DNA polymerase 7 (EC 2.7.7.7), probably synthesizes the DNA of these organelles [ 12--15]. The biological function of the third mammalian DNA polymerase, DNA polymerase fl (EC 2.7.7.7), is u n k n o w n and is usually considered to be involved in DNA repair [1--4]. The most direct evidences to this effect come from data of Bertazzoni et al. [16], and Waser et al. [17]. In searching for a function of DNA polymerase fl, we previously found that it is 6--30 times more efficient than DNA polymerase a in replication of DNA containing unrepaired lesions [ 18]. Similar results have earlier been reported by Coetzee et al. [19], who showed that DNA polymerase fi specifically replicates DNA damaged by bleomycin in vitro. In this paper the abilities of DNA polymerases a and fl to replicate another damaged template are examined. This was DNA irradiated in vitro with ultraviolet light treated with Micrococcus luteus correndonuclease [20]. Such a treatment introduces single-strand incisions in the vicinity of the pyrimidine dimers and generates 3'OH and 5'P termini [21]. Ultraviolet-irradiated, correndonuclease incised, DNA stimulates the nick translation reaction of bacterial DNA polymerase I, which leads to excision of the regions containing dimers, and to filling of gaps [22]. Animal DNA polymerases a and ~ do n o t contain 5' -~ 3'-exonuclease activity, and addition of dNTPs to 3'OH ends generated by correndonuclease could be possible only because the secondary structure of DNA in the vicinity of pyrimidine dimer is disturbed. Demonstration of the DNA polymerase reaction with a template in which the replicated region is partially overlapped by a nonexcised dimercontaining region would indicate the mechanism according to which the 'patch and cut' excision repair could proceed in animal cells. Materials

Reagents. Deoxyribonucleoside-5'-triphosphates (Sigma Chem. Co.) were additionally purified on Whatman DEAE-cellulose. [3H]dTTP was purchased from Radiochemical Centre, Amersham, U.K. [3H]Thymidine was from UVVR, Czechoslovakia; Agarose was from BDH; CsC1, from Serva. Activated DNA in 20 mM KC1 was prepared as in Ref. 23. Sonicated calf t h y m u s DNA in 30 mM NaC1 plus 3 mM Tris-HC1 (pH 7) was obtained as in [18]. Supercoiled Col E1 DNA and 3H-labeled Col E1 DNA of Escherichia coli JC 411 (Col E l ) cells were isolated according to Clewell et al. [24] and contained less than 5% of the open circular form. The final products were dissolved

248 in 50 mM Tris-HC1 (pH 8.0), 5 mM EDTA and 50 mM NaC1. 3H-labeled E. coli DNA (106 dpm/~g) of E. coli 15 T- cells was isolated by centrifugation in a CsC1 gradient [25,26]. DNA polymerase a from calf thymus was isolated according to Fansler et al. and Bollum et al. [23,27,28] as described [18] and dissolved in 50 mM KP i with 1 mM mercaptoethanol having the specific activity 10 000 U/mg protein. DNA polymerase ~ of calf t h y m u s was isolated according to Chang [ 29], additionally purified on a second DNA-cellulose column and dissolved in 0.2 M NaC1, 0.02 M Tris-HC1 (pH 8.0) 1 mM mercaptoethanol, 0.1 mM EDTA, and 50% glycerol having the specific activity 40 000 U/mg protein. One unit of DNA polymerase incorporates 1 nmol of dTMP into the activated DNA during 60 min at 37°C. According to the assay of exonucleases [18], their activity in DNA polymerases ~ and ~ did n o t exceed 1% of the polymerization activity. According to the assay of endonucleases [ 18] using 0.5 pg/ 50 pl PM2 DNA (a kind gift of P. Naimski, Institute of Biochemistry and Biophysics, Poland), 12.5 and 1.5 units of DNA polymerases a and ~, respectively, introduced 0.3 and 0.1 incision per DNA molecule during 3 h at 37°C. Correndonuclease from M. luteus, the fraction after Sephadex G-75 (in 100 mM KPO4, pH 7.6, 5 mM mercaptoethanol, 5 mM EDTA) containing 1500 U/ml [20], was kindly provided by Dr. L. Grossman, The Johns Hopkins University, U.S.A. One unit of correndonuclease was defined as the activity which incises 10 pmol of pyrimidine dimers during 30 min at 37 °. Correndonuclease was over 97% specific for ultraviolet-irradiated DNA and had less than 3% of contaminating endonuclease activities [20]. DNAase I was purchased from Worthington. Methods Ultraviolet irradiation. DNA (90 to 500 pg/ml) was irradiated in a 1-mm pathlength cuvette with a low pressure 15 W mercury lamp (British Thermal Syndicate) emitting mainly at 254 nm. An acetic acid/water (2 : 1) filter, cutting off irradiation below 230 nm was used. The incident fluence rate was 4.1 J/m2/s as measured by photolysis of a 1 0 - 3 M dimethyluracil, the quantum yield for which was 0.006 [30]. Assay for repair exonucleases. Native 3H-labeled E. coli DNA contained only 0.06% of the acid-soluble ~H-labeled material. This DNA and the same preparation ultraviolet-irradiated with a dose of 400 J/m 2, were incubated with correndonuclease and DNA polymerase ~ under conditions described in Fig. lB. The label precursor was omitted. After 2 h reaction the radioactivity of acid-soluble material was measured in Bray scintillator [18]. The acid-soluble 3H-labeled fraction obtained after the exhaustive digestion with DNAase I represented 100% of 3H-labeled DNA. The a m o u n t of nucleotides generated by repair exonucleases was calculated by subtracting the acid-soluble 3H-labeled material obtained for native DNA from that obtained for irradiated DNA. The reaction with D N A polyrnerase ~. Sonicated calf t h y m u s DNA or Col E1 were ultraviolet-irradiated, treated with correndonuclease as given in Table I, Expt. 1 and incubated with 5.0 units of DNA polymerase ~ for 2 h at 37°C. The reaction mixtures contained also in a final volume of 60 ~1:17 mM phos-

249 600

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A

u 400 L

& o

200

C:

13.. I.-'ID "l-

0 rn.fffl"[[~

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5

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15

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5

25

15

I

5

25

I

15

I

25

12 000

~8

D

000

4 000

o

f

m

I

I ,

Z.

8

I ,

12 Gel

,

,

I

I

. . . . . . . . .

I

i

•

/-

8

~ice

number

12

Z.,

8

12

Fig. 1. R e a c t i o n s of D N A p o l y m e r a s e fl w i t h n a t i v e a n d u l t r a v i o l e t - i r r a d i a t e d ( 6 0 J / m 2) C o l E1 D N A i n d u c e d b y n o n s p e c i f i c , D N A a s e I- o r c o r r e n d o n u c l e a s e - c a u s e d incisions. A - - C , [ 3 H ] d T M P i n c o r p o r a t i o n i n t o u n l a b e l e d t e m p l a t e s . D - - F , b r e a k d o w n of 3 H - l a b e l e d C o l E1 D N A t e m p l a t e u n d e r c o n d i t i o n s o f e n z y m i c r e a c t i o n . 3 0 pl (2.6 pg) o f n a t i v e C o l E1 D N A (A, C) o r 3 H - l a b e l e d C o l E1 D N A (D, F), a n d irrad i a t e d C o l E1 D N A (B) o r 3 H - l a b e l e d C o l E1 D N A (E) w e r e i n c u b a t e d f o r 2 b at 3 7 ° C w i t h 5 pl o f corr e n d o n u c l e a s e , 0.6 u n i t o f D N A p o l y m e r a s e /3 a n d w i t h o u t (A, B, D, E) or w i t h 0.1 p g / m l D N A a s e I (C, F). T h e r e a c t i o n m i x t u r e s c o n t a i n e d also in a final v o l u m e o f 6 0 #1: S m M MgC12, 1.3 m M d i t h i o t h r e i t o l , 80 ~M d A T P , d G T P , d C T P a n d d T T P ( D - - F ) or [ 3 H ] d T T P ( 1 1 0 0 d p m / p m o l ) ( A - - C ) . T h e m i x t u r e s w e r e a n a l y z e d b y 1% agarose-gel e l e c t r o p h o r e s i s in t h e b u f f e r c o n t a i n i n g 4 0 m M Tris base, 20 m M s o d i u m acet a t e , 2 m M E D T A , a n d glacial a c e t i c acid a d d e d t o p H 8.2, for 18 h at 20 V a n d r o o m t e m p e r a t u r e . A f t e r w a s h i n g a n d s t a i n i n g w i t h 0 . 5 # g / m l e t h i d i u m b r o m i d e t h e s u p e r c o i l e d , o p e n c i r c u l a r a n d linear f o r m s o f C o l E1 D N A w e r e l o c a l i z e d o p t i c a l l y , as s h o w n s c h e m a t i c a l l y u n d e r e a c h d i a g r a m . Gels w e r e c u t i n t o 2 - r a m t h i c k slides, a n d t h e r a d i o a c t i v i t y was c o u n t e d in 10 m l o f Bray scintillator. C o u n t i n g e f f i c i e n c y f o r 3 H - l a b e l e d C o l E1 D N A ( 0 . 0 0 1 - - 3 ~g) p r e s e n t in t h e slices, c a l c u l a t e d a g a i n s t t h e c o u n t i n g e f f i c i e n c y f o r D N A o n W h a t m a n G F / C glass filters, was 84%. R e l a t i v e c o n c e n t r a t i o n s o f D N A are given in T a b l e I. A r r o w i n d i c a t e s t h e d i r e c t i o n of e l e c t r o p h o r e s i s .

phate buffer (pH 7.2), 1.3 mM dithiothreitol, 7 mM MgC12, 80 pM dATP, dCTP, dGTP and [3H]dTTP (1100 dpm/pmol). An acid-precipitable 3H-labeled material was counted as in Ref. 18. Results To demonstrate the abilities of DNA polymerase ~.and/3 to replicate DNA vis-&-vis correndonuclease incision, the reactions of DNA polymerases with the following DNA preparations were compared: (1)ultraviolet-irradiated Col E1 DNA incised with correndonuclease, (2) native Col E1 DNA, and (3) native Col E1 DNA nicked with DNAase I.

250 TABLE LIMITED

I INCISION

OF Col E1 DNA BY CORRENDONUCLEASE

OF DNAase

I

U n l a b e l e d o r 3 H - l a b e l e d Col E 1 D N A w a s i r r a d i a t e d w i t h u l t r a v i o l e t i r r a d i a t i o n as g i v e n i n t h e t a b l e a n d d e s c r i b e d in M e t h o d s . I n E x p t . 1, 1 0 #1 o f t h e s e D N A s ( 3 p g ) a n d 5 pl o f c o r r e n d o n u c l e a s e w e r e i n c u b ated for 1 h at 37°C. The reaction mixtures contained also in a final volume of 30 pl: 10 mM Tris'-HCl ( p H 7 . 5 ) , 1 m M E D T A a n d 2 4 m M NaC1. N e x t t h e m i x t u r e s w e r e i n c u b a t e d w i t h 0 . 6 u n i t o f D N A o o l y m e r a s e fl f o r 2 h a t 3 7 ° C . T h e r e a c t i o n m i x t u r e s c o n t a i n e d a l s o i n a f i n a l v o l u m e o f 6 0 p l : 3 3 m M T r i s - H C l b u f f e r ( p H 8 . 5 ) , 1 . 3 m M d i t h i n t h r e i t o l 7 m M M g C l 2 , 0 . 1 M NaC1, S 0 p M d A T P , d G T P a n d [ 3 H ] dTTP (1100 dpm/pmol). The enzymic reaction and agarose-gel electrophoresis of Expt. 2 are described in F i g . 1. C o n c e n t r a t i o n s of the supercoiled (CCC), open circular (OC) and linear (L) forms of DNAs were evaluated optically or by cutting gels and counting of the 3H-labeled material. Results of optical evaluat i o n o f 3 H - l a b e l e d C o l E1 D N A c o n c e n t r a t i o n s (not shown) were the same as those for unlabeled DNA. DNA preparations in Expt. 1 and 2 were isolated separately; this can explain differences in their stabilities. Expt.

1

DNA

Col E1 DNA

Enzymes in reaction mixture

Ultraviolet dose (J/m 2 )

CCC

OC

L

0 60 400 2000

50 5 0 0

50 90 50 2

--

0 60

20 20

80 80

0 60

28 1

70 90

2 9

0

2

90

8

--

0 60

21 24

79 76

Correndonuclease, P o l y m e r a s e fl

0 60

15 0.5

83 90

2 9.5

Correndonuclease, P o l y m e r a s e ~, DNAase I

0

1

90

9

Correndonuclease, P o l y m e r a s e fi

Col E 1 D N A

Correndonuclease, P o l y m e r a s e /3 Correndonuclease, P o l y m e r a s e ~, DNAase I 2

3 H-labeled Col E 1 D N A

Form of DNA (%)

5 50 2

The ultraviolet irradiation-dose and conditions for the correndonuclease and DNAase I reactions were established, enabling measurements of DNA polymerase product and incision number. The DNA polymerase reaction was followed by the [3H]dTMP incorporation into unlabeled Col E1 DNA. The amount of incisions was estimated in parallel samples with all-labeled Col E1 DNA and unlabeled dTTP. The average incision number per DNA molecule was calculated from the fraction of electrophoretically separated superhelical form in the total 3H-labeled Col E1 DNA molecule population, applying the Poisson distribution. The assumption was made that the incision numbers in labeled and unlabeled DNA were the same. Optical evaluation of the DNA bands in the agarose gels supported this assumption (Figs. 1 and 2).

Reaction o f DNA polymerase fl with nonspecifically incised Col E1 DNA Data in Table I and Fig. 1 show that under the applied conditions a part of

251

B B

C C A

D D F F E E

G H I J

Fig. 2. A g a r o s e - g e l e l e c t r o p h o r e s i s o f n a t i v e o r u l t r a v i o l e t - i r r a d i a t e d Col E1 D N A t r e a t e d w i t h c o r r e n d o n u c l e a s e plus D N A p o l y m e r a s e / 3 , w i t h o r w i t h o u t D N A a s e I. R e a c t i o n m i x t u r e s A - - F , t h e s a m e as in Fig. 1, a n d m i x t u r e s w i t h o u t e n z y m e s , c o n t a i n i n g n a t i v e ( G ) o r i r r a d i a t e d ( 6 0 J / m 2) ( H ) Col E1 D N A o r n a t i v e (I) o r i r r a d i a t e d (J) 3 H - l a b e l e d Col E1 D N A , w e r e i n c u b a t e d a n d a n a l y s e d b y e l e c t r o p h o r e s i s as d e s c r i b e d in Fig. 1. T h e D N A b a n d s w e r e p h o t o g r a p h e d using a filter w h i c h c u t o f f r a d i a t i o n b e l o w 5 6 0 nm.

the superhelical form of Col E1 DNA was converted to the open circular form. The extent of this conversion was the same, or only a little greater, in the incubation mixture with unirradiated Col E1 DNA containing correndonuclease and DNA polymerase fl (Table I, Fig. 1D). These nonspecific incisions were probably due to the action of u n k n o w n nucleases contaminating the system and some spontaneous hydrolysis of the phosphodiester bonds. The average number of the nonspecific incisions, calculated for unirradiated 3H-labeled Col E1 DNA incubated with correndonuclease and DNA polymerase/3, a m o u n t e d in three experiments to 1.9, 1.0 and 1.7 per DNA molecule, respectively (Table II). In three experiments the number of [3H]dTMP molecules incorporated by

252 TABLE

II

NUMBER OF dTMP MOLECULES INCORPORATED BY DNA CIRCULAR Col E1 DNA 3'-OH END FORMED BY NONSPECIFIC, DNAase I INCISION

POLYMERASE fl P E R CORRENDONUCLEASE

OPEN AND

E x p t . I I is a l s o d e s c r i b e d i n T a b l e I, F i g s . 1 a n d 2.

Expt.

Kind of incision

Average incision number per molecule

Amount of [3H]dTMP incorporated into Col E1 DNA (pmoles)

dTMP molecule number per incision

2

Nonspecific DNAase I Correndonuclease

1.9 a 2.6 a 3.3 a

0.5 0.6 c 2.7 c

0.4 0.4 1.3

3

Nonspecific Correndonuclease

1.0 a 3.2 b

0.2 4.0 c

0.3 2.0

4

Nonspecific DNAase I

1.7 a 4.1 a

0.2 0.5 c

0.2 0.2

Incision number was calculated. a Incision number was calculated from 3H-labeled DNA distribution. b Incision number was calculated from the number of dimers. c Value corrected by subtraction of the value equivalent to the nonspecific

incisions.

DNA polymerase fl per one nonspecific incision, as estimated with unlabeled Col E1 DNA (Fig. 1A), was 0.4, 0.3 and 0.2 respectively (Table II).

Reaction o f D N A polymerase fl with Col E1 D N A incised by DNAase I Conditions for limited hydrolysis of unirradiated Col E1 DNA with DNAase I are described in Fig. 1. Addition of correndonuclease, DNA polymerase fi and of its substrates to the reaction mixture ensured the s.ame number of nonspecific incisions in all samples studied. The average DNAase I incision number per one DNA molecule was calculated from the superhelical 3H-labeled Col E1 DNA fraction (Fig. 1F, Table I) after subtraction of the number of nonspecific incisions, measured in a parallel sample without DNAase I (Fig. 1D, Table I). In the experiments shown in Table II, 4.1 and 2.6 DNAase I-incisions per DNA molecule were obtained. The number of nucleotides incorporated per one DNAase I incision was calculated from [3H]dTMP incorporation into open circular Col E1 DNA (Fig. 1C) after subtraction of the a m o u n t of [3H]dTMP incorporated as a result of nonspecific incisions (Fig. 1A). For the two experiments presented in Table II these were 0.2 and 0.4 incorporated dTMP molecules per DNAase I-incision, respectively. As mammalian DNA polymerases are not stimulated by DNA carefully nicked by DNAase I [3], [3H]dTMP incorporation found may be regarded as a reflection of slight exonuclease contamination. Reaction o f D N A polymerase fl with correndonuclease-incised Col E1 D N A Results of the DNA polymerase reaction with Col E1 DNA irradiated with increasing ultraviolet irradiation-doses of 60, 400 and 2000 J/m 2, and incised by M. luteus correndonuclease, are shown in Fig. 3 and Table I. In Expt. 1,

253 2000

),

B

1200

400 rrh o

20 25 30 35

20 25 30 35 o

2000

~r.~1200 I ~00 ,i

~

i

20 25 30 35 Gel

slice

,i

i

20 25 30 35 40 number

Fig. 3. R e a c t i o n o f D N A p o l y m e r a s e fl w i t h C o l E1 D N A i r r a d i a t e d w i t h i n c r e a s i n g u l t r a v i o l e t d o s e s a n d i n c i s e d w i t h c o r r e n d o n u c l e a s e . C o l E1 D N A i r r a d i a t e d w i t h u l t r a v i o l e t at a d o s e o f 0 (A), 6 0 (B), 4 0 0 (C) o r 2 0 0 0 (D) J / m 2 w a s t r e a t e d w i t h c o r r e n d o n u c l e a s e a n d D N A p o l y m e r a s e ~ as d e s c r i b e d in T a b l e I, E x p t . 1. O n e h a l f o f e a c h r e a c t i o n m i x t u r e w a s a n a l y s e d b y a g a r o s e - g e l e l e c t r o p h o r e s i s as d e s c r i b e d in Fig. 1 a n d C o l E1 D N A w a s l o c a l i z e d o p t i c a l l y . T h e s c h e m a t i c d r a w i n g s u n d e r e a c h d i a g r a m s h o w D N A l o c a l i z a t i o n , w h i l e t h e relative c o n c e n t r a t i o n s o f D N A are g i v e n in T a b l e I ( E x p t . 1). A r r o w i n d i c a t e s t h e direction of electrophoresis.

conversion of unirradiated superhelical Col E1 DNA to the open circular form by 50% was due to nonspecific incisions described earlier. Col E1 DNA irradiated with a dose of 60 J/m: was converted to the open circular form to the extent of 90%. In addition to the small amounts of the linear form, about 5% of the superhelical form was still present in the electrophoretic gel. At 400 J/m 2 further DNA degradation occurred and appearance of two almost equally intense DNA bands of the open circular and linear forms were observed. At the highest ultraviolet-dose of 2000 J/m 2 only homogeneous DNA bands of the open circular and linear forms were present but hardly visible, and the DNA degradation products were spread within the agarose gel. Fig. 3 shows that, according to expectations, no [3H]dTMP incorporation was found in the superhelical Col E1 DNA band, whereas it was present in the fractions containing the open circular and linear forms. In case of Col E1 DNA irradiated with a dose of 2000 J/m 2, for most electrophoretic fractions the counts were higher than for the background (Fig. 3D), probably resulting from the DNA polymerase reaction with products of Col E1 DNA degradation. The dose of 60 J/m 2, giving a relatively large amount of the incised open circular form, and a still measureable amount of the superhelical form (Table I) was applied in further experiments. Results of the reaction of correndonuclease and DNA polymerase fl with Col E1 DNA and 3H-labeled Col E1 DNA, both irradiated with a ultraviolet irradia-

254 tion-dose of 60 J / m 2, are shown in Fig. 1B, E and Table I, Expt. 2. The total number of incisions, calculated for irradiated 3H-labeled Col E1 DNA (Fig. 1E), was 5.2 per DNA molecule. Subtraction, from this value, of the number of nonspecific incisions (Fig. 1D) gave the average number of correndonuclease inci~ sions, i.e. 3.3 per DNA molecule (Table II). The a m o u n t of nucleotides incorporated per one correndonuclease incision was calculated from [ 3H]dTMP incorporation (Fig. 1B), after subraction of the a m o u n t of [3H]dTMP incorporated due to nonspecific incisions (Fig. 1A). As given in Table II, DNA polymerase fi incorporated 1.3 dTNP molecule per one correndonuclease incision. In another experiment shown in Table II the correndonuclease incision number was calculated on the assumption that it equals the pyrimidine dimer number (3.2) calculated for a dose of 60 J/m 2 [31]; in this case a very similar value, i.e. 2.0 dTMP molecule incorporated per one correndonuclease incision, was obtained (Table II, Expt. 3). The present results show that, though the DNA polymerase fi reaction induced by ultraviolet-irradiated DNA incised by correndonuclease is very discrete, it is distinctly more intense than that with nonspecific or DNAase I-incised DNA.

Determination o f repair exonucleases splitting o f ultraviolet-irradiated D N A incised by correndonuclease All enzymes used were acceptable with respect to contamination with endoand exonucleases. However, t h e y were not tested for repair 5' -~ 3' exonuclease which could excise a dimer-containing DNA fragment [32]. Thus the additional assay measuring the a m o u n t of nucleotides splitted from DNA ends formed by correndonuclease was performed. For three experiments with E. coli DNA irradiated with a doge 400 J / m 2, the amounts were 1.8, 2.2 and.2.4 pmol. These values were 30 times lower than the a m o u n t of four dNMPs incorporated into the same irradiated (400 J/m 2) DNA in a parallel sample (59 pmol). They were also distinctly lower than the a m o u n t of four dNMPs incorporated by the same enzyme into irradiated (60 J/m 2) Col E1 DNA (Table II). Requirements o f the D N A polymerase {J reaction The reaction was performed under conditions described in Table I, Expt. 1 and followed by counting the acid-precipitable 3H-labeled material. Incorporation of [3H]dTMP into irradiated (60 J/m 2) and correndonuclease-treated calf t h y m u s DNA was reduced to 20% when three dNMPs were omitted in the reaction mixture. There was no reaction in the absence of Mg2÷. At the presence of two inhibitors of DNA polymerase a, i.e. mercuribenzoate and N-ethylmaleimide [1--3] [3H]dTMP incorporation a m o u n t e d to 105 and 80% of that in a control sample. Lack o f reaction with D N A polymerase a The reaction of DNA polymerase a with native and ultraviolet-irradiated DNA, both treated with correndonuclease, was studied as described in Methods. [3H]dTMP incorporations into native sonicated calf t h y m u s DNA (23 pmol) or Col E1 DNA (1.1 pmol) were equal to [3H]dTMP incorporation into

255 the above DNA preparations irradiated with a dose of 60 J/m 2 (23 and 1.5 pmol, respectively). Even at a dose of 1400 J/m 2, when the number of dimers is a b o u t 20 times greater and double-strand breaks of DNA helix are possible (compare Fig. 3), the difference in the DNA polymerase a reaction between native and irradiated calf thymus DNA was negligible (7 pmol). Discussion The comparison of the amount of dTMP incorporated per one correndonuclease incision with that incorporated per one DNAase I incision demonstrated that DNA polymerase fl can replicate short template partially overlapped by non-excised DNA strand. L o w dTMP incorporation per DNAase I incision is consistent with the characteristics of mammalian DNA polymerases [3]; these enzymes require a singlestranded template, whereas interchain bonds in the vicinity of a DNAase I nick are not disrupted. In contrast, pyrimidine dimers cause disruption of hydrogen bond in ultraviolet-irradiated DNA [21]. This distortion of the DNA secondary structure has appeared to be sufficient for DNA polymerase fl to incorporate dTMP. Assuming that per each thymidylic residue three other nucleotides were incorporated, the length of the obtained DNA polymerase fl product was calculated to be 5--8 nucleotides. This value is consistent with the reported length of the singlestranded DNA region resulting from one pyrimidine dimer formation [33] and one correndonuclease incision [ 21]. In contrast to DNA polymerase fl, no reaction between DNA polymerase a and ultraviolet-irradiated, correndonuclease-incised DNA was found. As in this DNA the gap is still overlapped by the non-excised DNA fragment, the result cannot be directly compared with the data of Bose et al. [9] and Fischer et al. [34], proving the ability and the inability, respectively, of this enzyme to fill small gaps in DNA. However, it support evidences on greater efficiency of DNA polymerase fl than a for in vitro replication of damaged DNA [18,19]. The applied DNA may be treated as a model for in vitro studies of the 'patch and cut' excision repair. If so, it indicates that the DNA polymerase fl reaction can precede the exonuclease action during DNA repair. Acknowledgments We thank Dr. L. Grossman for his generous gift of M. luteus correndonuclease, Dr. A. Soltyk for Col E1 DNA and Mr. P. Naimski for PM2 DNA. We are grateful to Dr. D. Shugar and Dr. A. Michalowski for their critical reading of the manuscript. This work was supported by grant No. 1302 from the Polish Governmental Research and Development Programme PR-6 'Control of Malignant Neoplasms' and by the Polish A c a d e m y of Sciences, Project 09.7.1. References 1 L o e b , L.A. ( 1 9 7 4 ) in T h e E n z y m e ( B o y e r , P.O., ed.), Vol. 10, pp. 1 7 4 - - 2 0 9 , A c a d e m i c Press, N e w York 2 Weissbach, A. ( 1 9 7 5 ) Cell 5, 1 0 1 - - 1 0 8

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