Eur. J. Biochem. 172, 155-159 (1988) 0 FEBS 1988

Excision of apurinic and/or apyrimidinic sites from DNA by nucleolytical enzymes from rat brain Vladimir A. IVANOV, Tatyana M. TRETYAK and Yury N. AFONIN Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino

(Received July l/October 19, 1987) - EJB 87 0758

Apurinic and/or apyrimidinic (AP) sites were excised from PM2 phage DNA by two enzymes: an AP endodeoxyribonuclease isolated from rat neocortex chromatin and a rat brain exodeoxyribonuclease, DNase B 111. The resulting gap was filled with DNA polymerase /3 prepared from rat liver and finally ligated by Escherichiu coli DNA ligase.

A repair of DNA damage in nondividing long-lived brain cells appears to be of prime importance for maintaining the functional integrity of a genome. The stability of the DNA repair machinery is expected to provide normal functioning of the mammalian central nervous system. Indeed there is a relationship between the magnitude of the defect in excision DNA repair pathways and neurological abnormalities in patients with genetic diseases [l - 31. The most common DNA damage is apurinic and/or apyrimidinic (AP) lesions. The AP lesions in DNA may result from spontaneous depurination [4], from exposure to different chemical and physical agents [5] or from the action of various DNA glycosylase activities, which remove altered bases from DNA [6]. Mammalian en- zymes, which can immediately participate in the processes of the excision DNA repair of AP sites, were found in rat liver [7- 3 11, HeLa cells [12], human placenta [13 - 151, human fibroblasts [16] and calf thymus [17], however, a set of ana- logous enzymes observed in the brain is very deficient [18,19]. Cerebral DNA polymerases and DNA ligases have only been studied sufficiently to shown that they are similar to those from other mammalian tissues [19]. At the same time, data suggest some specificity of DNA repair in cerebral cells [20 - 221 which may be due to a short DNA repeat length of chroma- tin nucleosomal structure in neurons, unique in higher eukaryotic cells [23, 241 and the loss of DNA polymerase a [25] involved in repair pathways of some DNA damage [26]. Tissue specificity of brain DNA repair suggests the presence in the brain of repair enzymes possessing some special proper-

Correspondence to V. A. Ivanov, Institut Biologicheskoj Fiziki, Akademiya Nauk SSSR, Pushchino, Moskovskaya Oblast', USSR- 142292

Part of this work was done at the Institute of Biochemistry and Physiology of Microorganisms, Academy of Sciences of the USSR, Pushchino.

Abbreviations. AP, apurinic and/or apyrimidinic; DNA I, native covalently closed circular duplex DNA; DNA I ', relaxed covalently closed circular duplex DNA; DNA 11, nicked circular duplex DNA; DNA 111, unit-length linear duplex DNA, ds, double-stranded; ss, single-stranded.

Enzymes. DNA polymerase j3 (EC 2.7.7.7); deoxyribonuclease I (EC 3.1.21.1); snake venom phosphodiesterase (EC 3.1.4.1); Escherichia coli DNA ligase (EC 6.5.1 . l ) ; bacterial alkaline phospha- tase (EC 3.1.3.1); T4 polynucleotide kinase (EC 2.7.1.78).

ties. We previously reported a specific exodeoxynuclease localized in neuronal cell nuclei from the rat brain and suggested a repair function for the enzyme [27]. Recently we have found an endodeoxyribonuclease in rat neocortex chromatin recognizing the AP sites in DNA [28].

In the present work we demonstrate an excision of AP sites from DNA by the brain enzymes in vitro. Since DNA polymerase p and DNA ligase I1 are also found in rat brain cells [25, 29, 301, all the activities used for the useful repair in vitro are thus present in the brain cells.

MATERIALS AND METHODS The enzymes

The exodeoxyribonuclease, DNase B 111, was isolated from rat brain and purified to electrophoretic homogeneity [27]. This DNase, localized predominantly in neuronal cell nuclei, is a 5' + 3'-directed exonuclease [31]. The standard assay mixture (buffer A) contained 50 mM Tris/HCl, pH 8.4, 1 mM dithiothreitol, 0.5 mM Mg2+, 5% glycerol, 170 pg/ ml crystalline albumin. One unit of the enzyme, as defined previously, catalyzes the release of 1 nmol acid-soluble nucleotide from poly(dA) in 20 min at 37°C.

The AP endodeoxyribonuclease has recently been found for the first time in rat neocortex chromatin [28]. In a prelimi- nary paper, we reported observation and partial purification of the enzyme. In the present work we use a highly purified preparation of the neocortex AP DNase. This enzyme prep- aration was obtained by successive chromatography on DEAE-cellulose, hydroxyapatite and DNA-agarose. The en- zyme has a pH optimum at 7.8, requires Mg2+ or Mn2+ as a cofactor and has a relative molecular mass of about 28000. The purification and the properties of the enzyme will be reported in detail in a subsequent paper. One unit of the AP DNase catalyzes the incision of DNA near 0.1 nmol AP sites in 10 min at 37°C.

DNA polymerase /3 was prepared from rat liver according to the procedure described by Chang [32]. The enzyme unit is as defined there. To determine the DNA polymerase j priming activity of AP PM2 DNA incised with neocortex AP endonuclease, the incised DNA was precipitated with ethanol an incubated at 37°C in a reaction mixture containing

156

100 mM Tris/HCl, pH 8.0, 5 pM [ce3'P]dTTP (10 pCi), 60 pM each of dATP, dGTP and dCTP, 20 mM Mg2+, 2 mM dithiothreitol, 0.5 mM Na2EDTA and DNA polymerase 1 (0.75 unit/pg DNA substrate). Aliquots (0.3 pg DNA) were removed at various times and the DNA was precipitated with cold 0.5 M perchloric acid in the presence of 30 pg Escherichia coli DNA. The precipitated material was collected on a Whatman filter, dried and counted in a liquid scintillation system. In the DNA repair experiments, the reaction mixture (buffer C) was the same, except that 60 pM dTTP was used instead of labeled dTTP.

E. coli DNA ligase, deoxyribonuclease I and bacterial alkaline phosphatase were obtained from Worthington Bio- chemicals, snake venom phosphodiesterase from Boehringer- Mannheim and T4 polynucleotide kinase from New England Biolabs.

DNA preparations

Supercoiled PM2 [3H]DNA (23 700 cpm/pg) was prepared as described previously [33]. It was stored at 119 pg/ml in 1 mM Tris/HCl, pH 7.5, 1 mM Na'EDTA. [3H]DNA, isolat- ed from E. coli, had a specific radioactivity of about 35000 cpm/pg. ssDNA was obtained as described previously [27]. Partial depurination of PM2 [3H]DNA was carried out at 70°C in 10 mM sodium citrate, pH 5.0 and 100 mM NaCl according to [4] for 5 - 15 min to generate between one and three AP sites per duplex DNA circle. To obtain the native PM2 [3H]DNA preparation with one nick per molecule, we used snake venom phosphodiesterase which possesses an in- trinsic endonuclease activity specific for DNA I of PM2 phage [34]. The reaction of the DNA with the phosphodiesterase was performed as described by Kowalski [35]. To incise partially depurinated PM2 [3H]DNA, it was incubated in buffer B (40 mM Tris/HCl, pH 7.8, 10 mM Mg", 1 mM dithio- threitol, 120 pg/ml crystalline albumin) with limiting concen- trations of the neocortex AP DNase at 37°C. 5'-32P-end- labelling of E. coli ssDNA and incised AP PM2 DNA was as previously described [36] with the exception that either E. coli ssDNA of AP PM2 DNA I1 were used instead of DNA restriction fragments.

Determination of the number of nicks, gaps and AP sites in PM2 DNA

To determine the average sum (N) of nicks, gaps and AP sites, alkaline treatment of DNA solutions was carried our according to [35]. We checked that the procedure hydrolyzes a phosphodiester bond near each AP sites coverting covalently a closed circular duplex DNA containing AP sites into nicked circular duplex DNA. Intact DNAs I and I" renature after heating and cooling at pH ~ 1 2 . 0 , whereas open forms of DNA (DNAs I1 and 111) do not. Lack of treatment allows one to detect only nicks and gaps (breaks). Aliquots (25 p1) were placed in the wells of 2-mm-thick agarose gels containing ethidium bromide (1.2% agarose; 1.5 pg ethidium bromide/ ml). Each well received 600 ng DNA. Electrophoresis was carried out as described previously [36] supplementing the running buffer with 1.5 pg/ml ethidium bromide. To measure the radioactivity in each band the procedure described by Goffin and Verly [9] was used. The average number N of breaks per DNA molecule was determined by Poisson analysis P71.

Table 1. Specificity of neocortex A P endodeoxyribonuclease The average number of nicks and gaps without (breaks) or with (N) AP sites per PM2 DNA molecule were determined after gel electrophoresis performed without or with a prior alkaline denatura- tion, respectively, as described in Materials and Methods. The assay samples containing PM2 DNA I (24 pg/ml) and neocortex AP DNase (75 units/ml) were incubated for 12min at 37°C in buffer B. The reaction was terminated by a 5-min heating at 65 "C in the presence of sodium dodecyl sulfate to prevent the action of the proteins on DNA migration. Controls were carried out without the enzyme. Par- tially depurinated DNA used as substrates contained an average of 1.07 AP site/molecule in experiment (11, 1.88 AP sites in experiment (2) and 3.22 AP sites in experiment (3)

DNA Controls Assay

N breaks breaks

Native - 0.21 0.23 Depurinated

expt 1 1.30 0.23 1.28 expt 2 2.15 0.27 2.11 expt 3 3.56 0.34 3.47

RESULTS

Absence of interfering endonucleases in the enzyme preparations and specificity of neocortex endodeoxyribonuclease for AP sites

Each enzyme preparation was incubated with PM2 [3H]DNA I under the conditions used in the repair exper- iments. The number of breaks and N after the incubation were determined by gel electrophoresis. For each enzyme prep- aration (neocortex AP DNase, DNase B 111, DNA polymerase /I, and E. coli DNA ligase) this number per molecule was low. The results are not presented separately since the controls in the respective experiments give the same information.

To investigate the specificity of neocortex AP endo- nuclease, PM2 DNA I was depurinated and incubated with the enzyme. Table 1 shows that the neocortex enzyme has no action on native DNA; its activity corresponds to the number of AP sites per substrate molecule.

Absence of exonuclease activity in preparation of neocortex AP endodeoxyribonuclease

E. coli [3H]DNA (3 pg) in 100 pl 10 mM NaCl, 1.0 mM sodium citrate, 10 mM Mg", pH 7.8, was mixed with 15 p1 of the same solution containing 0.005 unit DNase I and incu- bated for 20 min at 37°C. The nuclease was then inactivated by heating for 10 min at 70°C or 100°C to obtain either ds or ss activated [3H]DNA, respectively. At this point, an acid- soluble radioactivity of 2% was measured. The activated [3H]DNA solution (100 pl) was mixed with an equal volume of buffer B containing 15 units neocortex AP endodeoxy- ribonuclease, and incubated at 37 "C. Aliquots were taken at various times up to 150 min to measure the acid-soluble radioactivity. The results were identical for ds or ss activated DNA : the acid-soluble radioactivity remained the same as for the unincubated substrates (data not shown).

DNase B 111 hydrolyzes incised AP PM2 DNA

Repair of AP DNA is initiated by introducing an ss nick on dsDNA to the immediate neighbour of the AP site [38].

157

- I I I I I

3000 - E a u - p 2000 4J RI h 0 .r( l0OC

N 0

0 20 40 60 Time ( m i n )

Fig. 1. DNase B III substrate specificity for PM2 DNA and E. coli SSDNA. DNase B I11 was incubated at 37°C in standard assay mixture with either (0) E. coli SS[~H]DNA or (0) incised AP PM2 [3H]DNA (10 nmol/ml) that had on the average 1.3 nick/molecule. At 0, 10,20, 40 and 60 min, 300-pl aliquots were withdrawn and extent of acid- soluble nucleotide release was determined by perchloric acid precipi- tation and liquid scintillation spectrometry. Each aliquots contained 0.4 unit enzyme

Therefore an essential ability of excising exonuclease is ini- tiation of degradation of dsDNA from intra-strand nicks. We previously found that DNase B 111, which is specific for ssDNA and not inhibited by AP sites, hydrolyzes dsDNA treated with DNase I [27]. Since the activated dsDNA may contain both nicks and gaps, it remained to be seen whether the enzyme can function from an intra-strand nick. With this in mind, we used here AP PM2 I3H]DNA with a single ss nick per molecule created by neocortex AP endonuclease. Fig. 1 shows that nicked AP MP2 [3H]DNA (about 1.3 nick per molecule) and the E. coli SS[~H]DNA have about the same amounts of acid-soluble radioactivity, which means that the enzyme hydrolyzes the DNA from both the terminals and the intra-strand nicks at the same rate. Consequently, DNase B 111 requires termini to initiate a hydrolysis; these termini may be provided either by an ss break in duplex DNA or by a free ss end, but not by a free ds end. Its specificity is reminiscent of that of DNase VIII, 5’ -+ 3’-directed exonuclease from human placental nuclei [15].

0 20 4 0 60 Time (min)

Fig. 2. Efficiency of incisions made by neocortex AP endonuclease to serve as DNA polymerase p primers. DNA incision reaction mixture was prepared as described in Materials and Methods. Partially depurinated PM2 [3H]DNA I (24 pg/ml) containing about 1 . 1 AP site/molecule was incubated with neocortex AP endonuclease (25 units/ml) for 20 min at 37°C (0). The incised DNA product (1.6 pg) was subsequently used as a substrate for DNA synthesis with 1.2 unit DNA polymerase p (see Materials and Methods). Native PM2 [3H]DNA 1 was incubated with snake venom phosphodiesterase under conditions to make one nick per supercoiled molecule (0). The reac- tion mixtures without any enzymes (A) are presented as a control. The average number of nicks per DNA genome, as determined by the gel electrophoresis, is given in parentheses

The DNA polymerase /3 priming activity of DNA incised with neocortex AP endonuclease

The priming ability of depurinated DNA treated with the neocortex AP endonuclease using DNA polymerase /3 was studied in order to characterize the nicks introduced in the supercoiled AP DNA by this DNase. As shown in Fig. 2, the nicked AP PM2 DNA served as an efficient primer for DNA polymerase 8, indicating that the cut sites possessed a free 3’-OH group. Undepurinated PM2 DNA I1 incised by snake venom phosphodiesterase was used as a marker of the priming activity. The DNA synthesis rate in this case was the same.

The AP site excision product

AP PM2 DNA I (about 1.1 AP site for molecule) was treated with neocortex AP endonuclease, then the 5’-phos- phate ends were labeled with 32P. Incubation of this substrate (100 nmol/ml) with DNase B I11 (3.0 unit/ml) in buffer A increased the acid-soluble 32P. Analysis of the acid-soluble fraction after a 20-min incubation at 37°C on DEAE- Sephadex A-25, as described previously [27], shows that most of the 32P is eluted with the mononucleotides (results not shown). This suggests that the AP site might be excised as deoxyribose 5‘-phosphate by hydrolysis of the phosphodiester bond on its 3’-side and not as deoxyribose S‘-phosphate covalently bound to one or two nucleotides, as is the case when the AP site is excised by the 5’-3‘-exonuclease acitivity of E. coli DNA polymerase I [32]. The result corresponds to that reported previously for the hydrolysis of undamaged DNA [27].

DNA B 111 activates DNA polymerase f l priming activity of DNA incised with neocortex AP endonuclease

To determine the effect of DNase B I11 activity on the DNA synthesis reaction, partially depurinated PM2 DNA I (about 1.1 AP site/molecule) was incised by neocortex AP endonuclease and used as a substrate for DNA polymerase with or without DNase B 111. In the absence of DNase B 111, a slight level of incorporation into DNA I1 was observed. DNase B I11 stimulated both the extent and the rate of DNA synthesis (Fig. 3). The data can be interpreted to suggest that DNA polymerase p is capable of performing a limited amount of strand-displacement synthesis with this substrate. The DNase B I11 would then promote more efficient synthesis by removing duplicate nucleotide material, so as to allow more extensive ‘nick-translational’ synthesis. A similar result was obtained by other investigators [12], who used HeLa ezymes: exodeoxyribonuclease, DNA polymerase f l and AP endo- nuclease.

158

, I I 10000 - -

E a 8000- 0

a - a, 6000-

0 20 40 60 T i m e ( rn in)

Fig. 3. Ability of DNase B 111 to stimulate DNA synthesis by DNA polymerase /3 from incisions made by neocortex AP endonuclease. In- cised AP PM2 [3H]DNA (15 pg/ml) was incubated with DNase B I11 (4 units/ml) for 20 min at 37°C in buffer A (0) . The control was carried out without the enzyme (0). After heat inactivation of the exonuclease (for 5 min at 65"C), the DNA produced from the reaction was then used as a substrate for DNA synthesis with DNA polymerase /3 (1 1 units/ml) (see Materials and Methods). The average number of nicks per DNA genome, as determined by the gel electrophoresis, is given in parentheses

Table 2. Excision of AP sitesfrom PM2 DNA AP PM2 [3H]DNA I was incubated with the neocortex AP endonuclease and then with DNase B 111. The excised DNA product was used as a substrate for DNA synthesis with DNA polymerase /3 and DNA ligase as described in the last section of Results. The average number of nicks and gaps or nicks, gaps and AP sites per PM2 DNA molecule were determined after gel electrophoresis performed without or with a prior alkaline denaturation, respectively, as described in Materials and Methods. The difference between the two results is defined as the average number of intact AP sites. Partially depurinated DNA used as substrates contained an average of 1.07 AP site/molecule in experiment I, 1.88 AP site in experiment I1 and 3.22 AP sites in experiment 111. Abbreviations used here: AP DNase, neocortex AP endodeoxyribonuclease; DNase B 111, brain 5'-3'-exodeoxyribo- nuclease; DNA pol, DNA polymerase /3 from rat liver; DNA lig, DNA ligase from E. coli; Nor breaks, average number per PM2 DNA molecule, of nicks, gaps and AP sites or nicks and gaps, respectively; AP int, intact AP site

Expt AP DNase DNApol DNA N Breaks APint DNase B I11 lig

I - + + + +

I1 - +

111 -

+

+ -

+ + -

- 1.30 0.23 1.07 - 1.34 1.28 0.06 + 1.20 0.52 0.68 - 1.54 1.54 0.00 + 0.42 0.39 0.03 - 2.15 0.27 1.88 + 0.66 0.61 0.05 - 3.56 0.34 3.22 + 1.23 1.09 0.14

Excision repair o j A P sites in PM2 DNA

Three independent experiments are presented in Table 2; they give the same quantitative results. The treatment of the AP PM2 [3H]DNA I with neocortex AP endonuclease was performed in buffer B as described in the legend to Table 1. The incised AP PM2 [3H]DNA was precipitated with ethanol

and incubated with DNase B 111 in buffer A as described in the legend to Fig. 3, except that the amount of DNase B I11 was decreased to 0.5 unit/ml. In several tubes, the reaction mixture containing four deoxynucleoside triphosphates (buffer C) supplemented with 0.40 mM ATP and E. coli DNA ligase (25 units/ml) with or without DNA polymerase p (11 units/ml) was incubated for 120 min at 15°C. The low tem- perature and removal of the AP site by 5'-3'-exonuclease prior to the treatment with DNA polymerase allowed us to avoid a possible strand-displacement during the polymerization step [lo]. The average number of nicks and gaps (breaks) or nicks, gaps and AP sites (N) per PM2 DNA molecule were deter- mined by gel electrophoresis performed without or with a prior alkaline denaturation, respectively, as described in Ma- terials and Methods. The difference between the two results was defined as the average number of intact AP sites per molecule that were present in the dsDNA. In Expt I the AP PM2 [3H]DNA incubated without any enzyme contained an average of 0.23 break and 1.07 intact AP site per molecule (see Table 2). Treatment with neocortex AP endonuclease hydrolysed a phosphodiester bond near most of the intact AP sites: their number decreased from 1.07 to 0.06. Incubation with E. coli DNA ligase sealed the incisions made by the neocortex AP endonuclease: the number of intact AP sites, which was 0.06/molecule after the neocortex AP endonuclease treatment and inactivation of this enzyme, was raised to 0.68 by the DNA ligase. Treatment with the DNase B 111, which widened into gaps most of the incisions made by the neocortex AP endonuclease, resulted in the disappearance of intact AP sites.

After incubating with the four enzymes only 0.03 intact AP site/molecule was left and the number of total lesions (N), which were almost exclusively breaks, was decreased from 1.30 to 0.42/molecule. The presented data are qualitatively similar to those obtained by Gofin and Verly [lo] with the enzymes from rat liver chromatin. The authors advance two different hypotheses which could set the limits of the repair. If the repair of breaks had an absolute priority, the remaining lesions should result from incompletely repaired AP sites. Following this hypothesis, the average number of intact AP sites that would have been repaired is: 1.07-0.03 -0.42 = 0.621 molecule. If it were the repair of intact AP sites that had an absolute priority, the average number of intact AP sites which would have been repaired is: 1.30-0.42-0.03 = 0.85/mol- ecule. Consequently, of the 1.07 intact AP site/molecule in PM2 DNA, 0.62-0.85 were repaired in this experiment.

Expts I1 and I11 lead to a similar conclusion: of the 1.88 and 3.22 intact AP sites/molecule that were in the substrates, 1.17- 1.41 and 1.85-2.19 were repaired, respectively (see Table 2).

DISCUSSION We previously reported that a rat brain exodeoxy-

ribonuclease, DNase B 111, localized predominantly in nuclei from permanently nondividing neuronal cells, is specific for ssDNA, hydrolyzes effectively ssDNA containing AP sites but is inactive against ultraviolet-irradiated DNA [27]. In addition, it was demonstrated that the DNase B I11 degrades DNA in the 5' --f 3' direction in a partially processive manner [31]. All this suggests that DNase B I11 may be involved in repair of neuronal DNA lesions, such as AP sites, whether they were generated by the action of DNA glycosylases, differ- ent chemical and physical agents or spontaneous depurina- tion.

We have recently found an endodeoxyribonuclease in rat neocortex chromatin which recognizes AP sites in DNA [28]. Mosbaugh and Linn distinguish two classes of AP endo- nucleases and suggest that the two classes of enzymes can work together to excise a deoxyribose 5’-phosphate residue from an AP site producing a one-nucleotide gap into dsDNA which will be filled by the following steps of excision repair [12, 161. It is not clear in mammalian cells, however, whether single-AP-site excision by AP endonucleases occurs in vivo since there are a variety of exonucleases able to initiate ex- onucleolytic hydrolysis at such sites. Furthermore, reinsertion by mammalian DNA polymerases requires more extensive ss regions for productive polymerization in order to reinsert normal nucleotides during repair replication [38]. Therefore the determination of the repair ability of the neocortex AP endonuclease is of much interest in studying the incision/ excision pathway in nerve cells.

In the present paper, additional analysis of DNase B I11 and neocortex AP endonuclease was performed. This is an important issue in view of the exciting possibility that the enzymes may be involved in the repair of DNA lesions in brain cells. We have found here that DNase B I11 specific for ssDNA is also active against incised AP PM2 DNA (Fig. 1). The enzyme may thus function from intra-strand nicks as the exodeoxyribonucleases, the excision function of which is assumed or has already been proved [38]. The specificity of DNase B I11 is similar to that of DNase VIII, a 5‘ + 3’ exonuclease from human placenta, as is the case when the placental DNase excises ultraviolet-irradiated dsDNA after incision by dimer-specific endonuclease [15]. The AP site ex- cision product by DNase B 111 is a mononucleotide as is the case when the AP site is excised by the DNase IV [Ill. It is shown that neocortex AP endonuclease is specific for AP sites in PM2 DNA I and under the experimental conditions described attacks no native substrate (Table 1). In additions, the DNA incised by the neocortex AP endonuclease serves as an efficient primer for DNA polymerase fi (Fig. 2). The results obtained suggest that the enzyme is a typical AP endonuclease which hydrolyzes the phosphodiester bond that is the immedi- ate neighbour of the AP site on its 5’-side, producing 3’-OH and 5’-phosphate ends, and thus provides DNA synthesis by mammalian DNA polymerases. The pretreatment of incised primer DNA by DNase B I11 is found to stimulate DNA synthesis with DNA polymerase a, suggesting that the 5‘-3‘ exonuclease removes the AP site together with duplicate nucleotide material and thus promotes more efficient DNA synthesis. The ability of DNA polymerase fi to fill the gaps was proved by nascent covalently closed circular duplex DNA (see Table 2).

Bacterial enzymes were able to repair in vitro DNA con- taining AP sites: endonuclease VI (the major bacterial AP endonuclease), DNA polymerase I and DNA ligase (from T4- injected E. colz] [39]. The repair of DNA containing AP sites by the enzyme extracted from rat liver chromatin [9, 101 and HeLa cells [12] has been reported. We present here the ability of excision of AP sites from DNA by nucleolytical enzymes isolated from rat brain: neocortex AP endonuclease and DNase B 111 (Table 2). The results of this work suggest that the nucleolytical enzymes may be involved in vivo in the orderly successive actions in excision of AP sites from brain cell DNA

159

where the further steps are catalyzed by other repair enzymes, DNA polymerase p[25] and DNA ligase I1 [29, 301 that were also detected in neuronal cells as major forms.

We thank Dr Anatolij Melnikov and Dr Alexander Solonin for technical assistance and discussion.

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