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Mutation Research, 63 (1979) 387--392 © Elsevier/North-Holland Biomedical Press

ENDONUCLEOLYTIC ACTIVITY FOR T-IRRADIATED DNA IN NORMAL AND ATAXIA TELANGIECTASIA FIBROBLAST CELL EXTRACTS

R.B. SHERIDAN III and P.C. HUANG *

Department of Biochemistry, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205 (U.S.A.)

(Received 21 February 1979) (Revision received 27 June 1979) (Accepted 30 July 1979)

Summary

The increased sensitivity of ataxia telangiectasia cells towards ionizing radia- tion may be related to their inability to incise DNA near sites of radiation- induced base damages. When compared to 3 unaffected controls, crude extracts from 5 lines of fibroblast cells derived from ataxia telangiectasia patients were capable of incising 7-irradiated DNA to the same extent as normal cells as determined in a nicking assay, using the circular replicative form of ¢PX174. However, the types of alterations introduced into DNA by T-irradiation could be distinguished from sites of base loss due to depurination or depyrimidina- tion and from sites of base modification by OSO4. The specific endonuclease involved was demonstrated to be distinct from the apurinic endonuclease by its rate of temperature inactivation.

Ataxia telangiectasia (Louis-Bar syndrome, abbreviated as AT) is a heredo- familial disease transmitted in humans as a single autosomal recessive trait. The clinical symptomatology is characterized by progressive cerebellar ataxia, pro- gressive oculocutaneous telangiectasias, chronic and progressive sinopulmonary infections, predisposition towards lymphoreticular neoplasms, chromosome instability, and extreme radiosensitivity [6]. No unifying concept has linked the variable immunological features with the more uniform symptoms of ataxia and telangiectasia nor with the severe sensitivity towards ionizing radiations. Much interest has been generated in the study of AT as a multisystemic genetic disorder resulting from an inborn error of metabolism attributable to a single gene defect with pleiotropic effects. Many of the clinical and cytogenetic

* T o w h o m t o s e n d c o r r e s p o n d e n c e .

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observations can be rationalized by assuming the genetic defect to be an inability for ataxics to adequately repair either spontaneously or ionizing radiation-induced damage in DNA.

This reduced repair capacity in ataxia cells may occur at two levels: ( 1 ) t h e repair of lesions which are manifested as DNA-strand breaks and (2) the repair of lesions not involving DNA-strand breakage. Reports from this and other laboratories have failed to detect a deficiency in either the rate or extent of repair of single-strand breaks in DNA at both high (10 krad) [4,9,10] and low doses (400 rad) [7] of ionizing radiation. More recently ataxia cells have been shown to repair double-strand breaks as efficiently as control cells [3]. Thus, barring a difference in the fidelity of repair, strand breakage does not appear to be the source of the reported radiosensitivity in ataxia telangiectasia. However, evidence has been presented suggesting that some ataxia cells may be defective in the removal of altered nitrogenous bases from DNA, specifically the incision step near the site of damage [4]. These experiments were performed by measuring a reduction in 7-irradiation-induced repair replication in 6 of 12 AT lines tested and by detection of a defective incision step in at least 2 cell lines.

In this s tudy the ability for crude extracts to incise 7-irradiated DNA was quanti tated by measuring the conversion of modified 4PX174 RFI DNA to the RFII form. The results show that the endonuclease(s) responsible for incision at sites altered by ~/-irradiation is present in crude extracts from ataxia telangiectasia cells.

Materials and methods

Cells were grown in MEM (GIBCO) supplemented with 20% fetal calf serum (GIBCO) and 1X antibiot ic/antimycotic (GIBCO) at 37°C in a humidified chamber with 5% CO2. Upon reaching confluency, 2 × 106 cells were trypsin- ized, washed twice with cold PBS by centrifuging and resuspended in 1 ml extraction buffer (100 mM NaC1, 50 mM Tris--HC1 pH 7.4, I mM trisodium citrate, 10 mM 2-mercaptoethanol and 20% glycerol) for sonication. Sonicates were stirred on ice for 60 min then centrifuged at 30 000 X g for 20 min. The supernatant was dialyzed for 16 h against a 500-fold volume of extraction buffer at 4°C and normally contained 1--2 mg/ml of protein as determined spectrophotometrical ly [8]. Prior to assay, the extract was diluted in 500 lzg/ml bovine serum albumin (BSA) containing 5 mM N-2-hydroxyethyl- piperazine-N'-2~thanesulfonic acid (HEPES) pH 8.0. Each reaction contained 1 nanomole irradiated tritiated 4PX174 RFI DNA, 5 mM MgC12, 25 mM HEPES pH 8.0, 5 ug BSA and 1 ag of crude protein extract in 40 ~l final volume. After incubation for 10 min at 37°C the reaction was stopped by the addition of two volumes of a mixture of 0.01% sodium dodecylsulfate (SDS) and 10 mM ethylenediamine tetraacetic acid (EDTA). To each tube was added 2 ml denaturation buffer (300 mM NaC1, 10 mM Na2HPO4, 25 mM EDTA pH 12.0) followed by neutralization with 0.4 ml 2 M Tris--HC1. Samples were collected on nitrocellulose filters, dried and counted by liquid scintillation. Procedures for the identification and quantitation of 4pX174 RFI and its conversion to RFII have been described earlier [8].

~/-Irradiation of (I)X174 RFI was performed with a 137Cs ~, source (Model

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M-38) giving a relative dose rate of 400 rad/min, when adjusted for a dose factor of 20%. The (PX174 DNA concentration was 100 nmol/ml in 50 mM NaC1, 1 mM Na2HPO4, pH 8.0. Apurinic DNA was prepared by heating qbX174 RFI in 10 mM NaC1, 10 mM trisodium citrate, 10 mM NaH2PO4, pH 5.0 at 70°C for 12 min. OsO4-treated DNA was made as follows. A solution of 0.4% OsO4 in deionized water and cPX174 RFI DNA in 2 mM Na2HPO4, pH 7.0 were raised to 70°C for 15 min. Next the two solutions were mixed in equal volumes to give a final concentration of 0.2% OsO4, 100 nmol DNA/ml, 1 mM Na~HPO4, pH 7.0. A further incubation of 15 min at 70°C was required for modification of the DNA. This was followed by 4 extractions with cold ether and by dialysis against 1 mM EDTA, 10 mM Tris, pH 8.0.

Alkaline hydrolysis of these various substrates was performed by incubation of the modified DNA's in 2 ml of denaturation buffer for 4 h at room tempera- ture. The solutions were neutralized with 0.4 ml 2 M Tris--HC1 and quanti tated as before.

Results

Under the conditions used here, two types of sites are introduced by 3'-irradiation. Class I sites are identified as single strand breaks (SSB) and are defined as damages which manifest themselves as strand breaks immediately upon alkaline denaturation. Class II sites are termed alkali-labile sites (ALS) and are defined by their slow conversion to single-strand scissions with prolonged incubation in alkali (4 h). Quantitating the relative numbers of sites using the Poisson distribution gave an approximate 1 : 1 ratio between SSB's and ALS's. Since our assay quantitates strand scissions, all class I sites will be measured in the initial background levels. If qbX174 RFI is irradiated until it contains an average one ALS per molecule the background will be 63% of the starting material (1 SSB/molecule), while just 23.5% of the original molecules will contain only ALS's. This high initial background (SSB) and the low sub- strate concentration (ALS) made rate of incision experiments virtually impos- sible to quantitate. However, extent of incision could be measured. As illustrated in Fig. 1, crude extracts are capable of incising all 3"-induced alkali- labile sites present. No new alkali-labile sites are created upon induction with extract and no enzyme-sensitive sites which are not also alkali-labile appear to have been introduced in the ¢PX174 circles under the chosen conditions of irra- diation. No activity was detected in boiled extract while the number of enzyme-sensitive sites exactly equalled the number of alkali-sensitive sites.

Fig. 2 summarizes the data for 3 control (open symbols) and 5 ataxia (closed symbols) cell lines in an extent of incision assay. No difference in the ability for ataxia to completely incise all sites present was observed. The line of unit slope represents one enzyme-sensitive site for every enzyme-sensitive site plus alkali-labile sites.

In Table 1, the rate constants of alkaline hydrolysis per modified site in • X174 RFI DNA are compared for several different treatments. The order of alkaline sensitivity is 3' > apurinic > OsO4 > unmodified. The data in Table 1 were calculated from the slope of the linear relation between the number of sites per molecule and the associated rate constants of hydrolysis as illustrated

390

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in Fig. 3. Different numbers of sites were introduced by varying the length of treatment time. The number of sites was determined from the Poisson distribu- tion, F = e -~ where F is the fraction of RFI surviving after complete alkaline hydrolysis and a is the number of sites per molecule. Rate constants were deter- mined from the relation F = e -kt where t is the time of alkaline hydrolysis for DNAs modified for increasing times and with various agents. Thus Fig. 3 represents the simple transformation a = k t . Since the rate of hydrolysis for unmodified DNA is so slow, the rate constant per site was determined from the intercept with the abscissa. The number of sites was estimated to be approxi- mately 10 772, the number of bases in a cPX174 RFI molecule.

In Fig. 4, a comparison between the temperature inactivation rates for the enzyme activities in crude extracts responsible for incision at apurinic and v-induced sites is shown. The measurements for both activities were made on aliquots from the same sample of crude extract (1 mg protein/ml) held at 50°C for increasing time. The results indicate that the apurinic activity is 8-fold more heat stable than the 7-endonuclease activity.

T A B L E 1

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Fig. 4. A c o m p a r i s o n b e t w e e n t h e t e m p e r a t u r e r e a c t i v a t i o n r a t e s f o r t h e e n z y m e a c t i v i t i e s m c r u d e e x t r a c t s r e s p o n s a b l e f o r i n c z s i o n at a p u r i n l c a n d T - r e d u c e d sates in D N A . A a p u r i n i c e n d o n u c l e a s e ; o, 9 ' - e n d o n u c l e a s e .

Discussion

On the basis o f measurements for extent of incision, all sites in the ~f-irradiated DNA that are recognized by the endonuclease in the cell extract are the same as those sensitive to alkaline hydrolysis. No new sites were detectable by incubating the DNA which had already been subjected to enzymatic incision in the extracts (Fig. 1) with alkali. This result suggests that N-glycosylases do not contribute significantly to those site-specific endo- nucleolytic reactions.

The data summarized in Fig. 2 show that crude extracts from both control and ataxia cells are capable of incising all 7-endonuclease-sensitive sites present from 0.1 to 1.2 ALS's per molecule as estimated from the Poisson distribution. This result was consistent with the demonstrated ability for CRL 1347, AT4BI, and AT5BI to undergo normal repair replication after ~-irradiation but was thought to be surprising in the case of CRL 1343 which has been shown to have reduced repair-replication presumably as a result of impaired incision [4] . To explain this descrepancy one must either assume the predominant lesions studied were different in each study or that CRL 1343's incision endonucleases can freely incise naked DNA at sites of radiation damages but are unable to recognize the same damage when the DNA is complexed with structural pro- teins such as histones in chromatin.

In order to characterize the ~ndonuclease -sens i t ive lesion, the predomi- nant radiation-induced damage was compared to two other DNA alterations (sites of base loss induced by heat depurination and 5,6-dihydroxy-5,6-dihydro- thymine introduced by modification with OsO4) for which normal incision has already been demonstrated in ataxia [5 ,8] . As shown in Table 1, the ALS's in

392

apurinic DNA are 6.3 times less stable than the OsO4 sites and 27 times more stable than the 7-induced sites. This difference in rate of alkaline hydrolysis suggests that all 3 forms of damage are chemically distinct lesions.

The data presented in Fig. 4 show that the 7-endonuclease is thermally inactivated at a rate 8 times as fast as the apurinic-site endonuclease. This suggests that two different enzyme activities are involved, each specific for its own lesion. In results not shown, we observed that these nucleases have differ- ent optimal requirements for magnesium at 5 and 15 mM for 7 and apurinic nuclease activities, respectively. Thus the 7-endonuclease shown to be present in crude extracts from ataxia cells is not only specific for 7-irradiated DNA but also is distinct from other known endonucleolytic activities.

Recently, cells from patients with ataxia were shown to contain normal levels of a, ~ and 7 DNA polymerases [1]. The successful excision of 5,6-dihydroxy-5,6-dihydrothymine from exogenous DNA by ataxia fibroblast sonicates in vitro suggests that the exonucleases responsible for excision are present [5]. Now, results from this s tudy show that the endonucleases responsible for incision at alkali-labile sites in 7-irradiated DNA are fully active in crude AT extracts. Thus the defect in those ataxia lines identified as repair- deficient may not be the lack of nucleolytic functions but a deficiency in the recognition functions needed to guide specific nucleases to the sites of base modification. One plausible explanation is that within the cell, recognition of the damage by the various nucleases may be inhibited by proteins associated with the DNA or there may be other enzymes that could be defective in AT cells that have not ye t been investigated.

Acknowledgments

Work done in this laboratory was supported in part by National Institutes of Health (R01 ES 01596 and P01 ES 00454).

References

i B e r t a z z o m , U., A. I . Scovassi , M. S t e f anmi , E. Glu lo t to , S. Spadar l and A.M. P e d n m , DNA poly- merases a , ~ and T m inher i t ed diseases af fec t ing DNA repair , Nucl . Acids Res. , 5 (1978) 2 1 8 9 - - 2 1 9 6

2 Georga tsos , J .G. , Acid phospha tases of h u m a n e r y t h r o c y t e s , Arch . B lochem. Blophys . , 110 (1965) 3 5 4 - - 3 5 6 .

3 L e h m a n n , A.R. , and S. Stevens , The p r o d u c t i o n and repai r of double s t rand breaks m cells f r o m n o r m a l h u m a n s and f r o m pa t ien ts wi th a taxla te langlectas la , Blochlm. Blophys. Acta , 474 (1977) 49 - -60 .

4 Pa terson , M.C., B.P. Smi th , P.A. Kn igh t and A.K. Ande r son , Atax ia te langiectas la an inher i ted h u m a n

disease revolving radiosens l t lv l ty , m a h g n a n c v , and defec t ive DNA repai r , m: A. Castel lam (Ed.) , Research m Pho t ob l o logy , 1977 , pp . 207 - -218 .

5 Rem sen , J .F. , and P.A. Cerut t i , Excis ion of g a m m a - r a y induced t h y m i n e lesions by p repa ra t ions f r o m a tax la te langiectas ia f lbroblasts , Muta t ion Res. , 43 ( 1 9 7 7 ) 1 3 9 - -1 4 6 .

6 Sedgwmk, R.P , and E. Boder , A tax ia te langlectas la , in: H a n d b o o k of Clinical Neu ro logy , 1972 , pp. 267 - -334 .

7 Sher idan I I I , R.B., and P.C. Huang , Single s t rand breakage and repai r in euka ryo t l c DNA as assayed by S 1 nuclease , Nucl . Acids Res., 4 (1977) 299 - -318 .

8 Sher idan I I I , R.B. , and P.C. Huang , A p u r i m c a n d / o r a p y r i m i d m i c endonuc lease ac t iv i ty in a tax ia te langmctas la cell ex t raots , Muta t ion Res. , 52 (1978) 129 - -1 3 6 .

9 Tay l o r , A.M.R. , D.G. H a r n d e n , C.F. Ar le t t , S.A H a n c o u r t , A.R. L e h m a n n , S. S tevens and B.A. Bridges, A t a x m te langlectas ia , a h u m a n m u t a t m n wi th a b n o r m a l r a d l a t m n sensi t ivi ty , Na tu re , ( L o n d o n ) , 258 (1975) 4 2 7 - - 4 2 9 .

10 Vincen t Jr . , R .A. , R.B. Sher idan I I I and P.C. Huang , DNA s t rand b reakage repa i r in a tax ia telangi- ectasia hb rob la s t -hke cells, M u t a h o n Res. , 33 (1975) 3 5 7 - - 3 6 6 .