Apurinic and/or apyrimidinic endonuclease activity in ataxia telangiectasia cell extracts
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Mutation Research, 52 (1978) 129--136 Elsevier/North-Holland Biomedical Press
APURINIC AND/OR APYRIMIDINIC ENDONUCLEASE ACTIVITY IN ATAXIA TELANGIECTASIA CELL EXTRACTS
RICHARD B. SHERIDAN III and P.C, HUANG
Department of Biochemistry, The Johns Hopkins University, School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, 21205 (U.S.A.)
(Received 20 January 1978) (Accepted 26 April 1978)
The possibility that the increased sensitivity of ataxia telangiectasia towards ionizing radiation is related to a DNA-repair deficiency has been examined fur- ther. When compared to unaffected controls, 6 lines of fibroblast cells derived from ataxia patients demonstrated a slightly reduced endonucleolytic activity (165 -+ 12 units vs. 214 + 28 units) towards apurinic and/or apyrimidinic sites as determined in a "nicking" assay.
Ataxia telangiectasia (Louis-Bar syndrome) is a human autosomal recessive disease which is associated with neurological degeneration, immunological defects and predisposition towards malignancy . A unique feature of this syndrome is its extreme sensitivity towards ionizing radiation. This was first observed clinically as a severe response to radiotherapy usually resulting in the death of the patient [3,6,16].
Because of interest in ataxia's apparent increased sensitivity towards ionizing radiation , an increasing amount of cytogenetic and biochemical informa- tion is being amassed. Upon exposure to ionizing radiation [1,20] or radio- imetic drugs such as methylmethane sulphonate and mitomycin C , a reduced capacity for survival of cultured cells derived from patients afflicted with ataxia was observed. These observations formed the basis for the hypo- thesis that ataxia telangiectasia is a disease associated with a defective repair mechanism for ionizing radiation induced damage in DNA.
Reduced repair capacity in ataxia cells may occur at two levels: (1) the repair of lesions which are manifested as DNA-strand breaks and (2) the repair
Abbreviations: BSA, bovine serum albumin; EDTA, etbylenediamine tetraacetic acid; HEPES, N-2-hydroxyethyloPiperazine-N'-2-ethanesulfonic acid; SDS, sodium dodecylsulfate.
of lesions not involving DNA-strand breakage. Reports from this and other laboratories have ruled out a deficiency in both the rate and extent of repair of single-stranded breaks in DNA at both high (10 krad) and low doses (400 rad) of ionizing radiation [19,21,24]. More recently ataxia cells have been shown to repair double-stranded breaks as efficiently as control cells . 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 ataxia cells may be defective in the removal of altered nitrogenous bases from DNA . These experiments were performed by measuring a reduction in 7-radiation-induced repair replication under anoxic conditions and by detection of an increased incision by crude extracts from Micrococcus luteus of "gamma endonuclease" sensitive sites remaining in DNA after whole cell repair incubations. At present, it is unclear what specific repair defect is responsible for these observations.
There are two known pathways of excision repair: (1) nucleotide excision- repair in which a piece of DNA containing the altered base is excised and (2) base excision-repair in which the modified base is removed either spon- taneously or enzymatically followed by incision at the apurinic and/or apyri- midinic site by an endonuclease . Since the apurinic endonuclease has been shown to incise both apurinic and apyrimidinic sites, it will hereafter be referred to as the Apu/Apy endonuclease . This study examines the second pathway and shows that the endonuclease responsible for incision at the site of base removal is present at slightly reduced levels in ataxia cells.
The rationale for investigating the levels of this endonucleolytic activity in ataxia cells is evident when one consideres the earlier work by cytogeneticists. The levels of spontaneous chromosome and chromatid aberrations in ataxia are high relative to unaffected controls; upon exposure to 7-rays or X-rays this difference is amplified [3,6,16]. Thus, if a defect in the repair of damaged DNA exists, one should probably look at lesions which can arise spontaneously as well as through irradiation. Work by Lindahl and Nyberg  demonstrates that depurination occurs spontaneously in DNA at physiological temperature, ionic strength and pH. Depyrimidation by a uracil N-glycosylase has been shown to occur as the result of misincorporation during replication . On the other hand, ionizing radiation could also cause both depurination and depyrimidation through modification of the four nitrogenase bases or by direct cleavage of N-glycosylic bonds . Since base loss occurs at a significant rate with or without the influence of radiation, the enzymes recognizing the apurinic and/or apyrimidinic sites are important ones to be examined.
Materials and methods
Fibroblasts were grown in 250 cm 2 culture flasks in MEM's supplemented with 20% fetal calf serum and 1X antibiotic/antimycotic at 37C in a humi- dified chamber with 5% CO:. Upon reaching confluency, 2 X 106 cells were trypsinized and washed twice with cold PBS. Cells were resuspended in 1 ml extraction buffer (100 mM NaC1, 50 mM Tris--HC1 pH 7.4, 1 mM trisodium citrate, 10 mM 2-mercaptoethanol and 20% glycerol) and sonicated. Extracts were stirred on ice for 60 min then centrifuged at 30 000 X g for 20 min. The
supernatant was dialzyed for 16 h against 500 ml of extraction buffer at 4 C. Protein content was routinely 1--2 mg/ml as determined spectrophoto- metrically . Prior to assay, extract was diluted in 500 mg/ml bovine serum albumin (BSA) containing 5mM N-2-hydroxyethyl-piperazine-N'-2-ethane- sulfonic acid (HEPES) pH 8.0. Each reaction mix contained I nanomole tri- tiated apX174 RFI with on the average 1 apurinic site per molecule, 15 mM MgC12, 25 mM HEPES pH 8.0, 5 pg BSA and between 0 and 1 #g of extract protein in 40 pl final volume. The reaction mixes were incubated for 10 min at 37C then stopped by the addition of two volumes of 0.01% sodium dodecyl- sulfate (SDS), 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. Activities were computed in the linear region of the assay. The acid phos- phatase assay was based on that of Georgatsos . The same extract was divided into two aliquots for these assays. Values represent averages for dupli- cate determinations. Substrates with increasing amounts of damage were used to measure the extent of incision by the Apu/Apy endonuclease under satu- rating enzyme conditions. These substrates were prepared by heating ~X174 RFI in 10 mM NaC1, 10 mM trisodium citrate, 10 mM NaH2PO4, pH 5.0 at 70C for various times. Assuming that the distribution of apurinic sites follows a Poisson distribution and that the probability of any site being nicked is iden- tical, then the average number of sites per DNA molecule is given by: F = e -a, where F is the fraction of molecules surviving after alkali treatment for 4 h to hydrolyze all apurinic sites and a is the average number of apurinic sites per molecule. Assays were performed at enzyme saturation with 1 nanomole DNA and 1 pg extract protein. Alkali-sensitive sites were measured by treating reac- tion mixes with denaturation buffer for 4 h before neutralization.
Control cell lines were obtained from Dr. George Thomas (GM37 and 38) and Dr. Edward Schneider (GRC 32). Ataxia cell lines AT4BI and AT5BI were kindly provided by Dr. Alan Lehmann, and the CRL lines were originally pur- chased from American Type Culture Collection. AT3RI and CRL1212 were designated as complementation group A and CRL1343 as B . Of all the lines shown to repair single-strand breakage normally , only CRL1343 and CRL1347 were included in this study.
We have employed a selective retention or "nicking" assay for the Apu/Apy endonuclease activity . Double-stranded, covalently closed, supertwisted X174 RFI DNA which has been modified at acid pH with heat to produce an RFI containing on the average one apurinic site was used. The modified DNA was incubated with the cell extracts under defined conditions. In the presence of the enzyme, the DNA is "nicked" at the site of the lesion forming the double-stranded open circle form RFII. Alkali is also capable of nicking at apurinic sites through a ~-elimination reaction. Upon denaturation followed by renaturation, nicked DNA circles undergo strand separation while intact circles snap back into double-stranded forms. When passed through a nitrocellulose
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MICROGRAMS OF CRUDE EXTRACT Fig. 1. The number o f (PX174 RF I c i rc les n icked per un i t t ime per #g o f cel l ex t rac t p ro te in as a measure o f the enzyme act iv i ty p resent in the var ious cell l ines. The un i t s o f enzyme present were measured in the l inear por t ion o f the curve
APU/APY ENDONUCLEASE AND ACID PHOSPHATASE MEASUREMENTS
Cel l l ines Apu/Apy endonuc lease un i t s Ac id phosphatase un i t s (pmol min -I #g-l) (pmol rain -1 #g-l)
Cont ro l s GM 37 187 26 GM 38 201 21 GRC 32 253 29
214 +- 28 25 + 3
Atax ia te lang iec tas ia CRL 1312 168 19 CRL 1343 163 19 CRL 1347 185 29 CRL 1346 145 24 AT 4 B I 170 23 AT 5 BI 157 20
165 -+ 12 22 4
t = 3 .18 t = 1 .08
P = 1 .6% P = 31 .8%
The levels o f Apu/Apy endonuc lease and ac id phosphatase in 3 cont ro l and 6 a tax ia l ines measured in un i t s o f p ieomoles o f subst ra te conver ted per ra in per pg o f c rude ext rac t p ro te in .
filter the single-stranded DNA is retained and the double-stranded DNA is not. The number of circles nicked per unit time per microgram of cell extract pro- tein is a measure of the enzyme activity present in the various cell lines. As seen in Fig. 1, an input of one nanomole of DNA with on the average one apurinic site per molecule and a reaction time of 10 min at 37C we find the reaction increases linearly between 0 and 0.4 pg of extract protein; however, beyond 0.4 pg the substrate becomes saturated. The sensitivity of this assay is best illustrated by the fact that only 200 cell equivalents of cell extract were needed to measure the units of enzyme present.
Table 1 summarizes results from the study of 3 control and 6 ataxia lines. Included as an internal control are the levels of acid phosphatase present in the cell extracts. Acid phophatase was chosen because its cellular function is pre- sumably unrelated to DNA repair. By applying a standard t-test we observed that there was no statistical difference between the levels of acid phosphatase for ataxia cells and controls but that there was a significant difference (40 units or a 22% reduction) between the levels of Apu/Apy endonuclease. However, our estimate suggests that even the lowest level of activity seen is far in excess of that needed to meet the normal requirements of the growing cell. Lindahl and Nyberg estimated spontaneous formation of apurinic sites to be 1.7--8.3 sites/cell/min. Assuming 2 X 103 cells give 1 pg of crude extract protein then 200 units of enzyme activity is equivalent to 6 X 106 apurinic sites nicked/ cell/min which is far in excess of the amount needed to metabolize all sites formed spontaneously.
The effect of the reduction in Apu/Apy endonuclease activity on the extent
, c i ~ | p
: ALKALI LABILE SITEi D ~ BOILED EXTRACT
0 = ENZYME SENSITIVE SITES = ENZYME SENSITIVE SITES + ALKALI LABILE SITES
3 6 9
TIME IN MINUTES OF DEPURINATION AT PHS.0, 70C
F ig . 2 . The extent of incis ion by the Apu/Apy endonuc lease under saturating enzyme condi t ions as a funct ion of increasing amounts o f damage in the substrate.
i00. o = GM38 = GM37 . ,o~o. . . * .~ . . _ -
90 o = GRC32 = CRL1347 ,A /
>~ 80 = CRL1343 ~ o > = AT4BI ~ o .
70 *= AT5BI ~ . = CRL1346 * y ,~"
60 d, o ~ - ~ /
0 1~ 20 3o 40 5o 6o 70 80 9o li0
ENZYME SENSITIVE SITES + ALKALI SENSITIVE SITES (% RFI SURVIVIN6)
Fig. 3. A summary of the extent of incis ion by 3 contro l and 5 ataxia cell l ines. The line of units slope represents one alkali sensit ive site for every enzyme-sens i t ive site.
of repair as a function of the amount of damage present was further investi- gated. DNA's with different levels of damage were prepared by heating at pH 5.0 for various lengths of time. The total number of apurinic sites induced were measured as a function of the percent unnicked circles surviving after alkali treatment. The number of apurinic sites cleaved at enzyme saturation by crude extract was also measured. In addition the number of apurinic sites not cleaved by the enzyme were measured by treating completed reaction mixes with alkali for 4 h. The results presented in Fig. 2 are what one would expect if for every alkali-sensitive site produced there is an enzyme molecule capable of nicking it, since one would expect to find no difference in the percent RFI surviving among all three treatments applied.
The data for 3 controls and 5 ataxia lines are summarized in Fig. 3. The line of unit slope represents one alkali-sensitive site for every enzyme-sensitive site. As the number of apurinic sites is increased, both control and ataxia extracts appear to be capable of incising all apurinic sites present. These results suggest that ataxia cells complete the incision step to the same extent as controls even with 22% less enzyme activity present.
Although the levels of Apu/Apy endonuclease in ataxia telangiectasia appear to be slightly reduced, we cannot detect an effect on the extent of repair due to this reduced activity. Thus on the basis of the evidence presented here we must conclude that a deficiency in base excision-repair at the level of the endo- nuclease specific for the site of base loss is probably not an explanation for the disease ataxia telangiectasia. However, as pointed out by Teebor and Duker  in their discussion of the levels of Apu/Apy endonuclease in Fanconi's anemia, multiple endonucleases of similar function may exist in the cell; there- fore, results from measurements with crude extracts may be of such a com-
posite nature that a defect in one of them may escape detection. In this sense, the observed reduction in activity with the ataxia cells may be a clue for more detailed analysis.