Mutation Research, 141 (1984) 41-44 41 Elsevier

MRLett 0594

Apurinic/apyrimidinic endonuclease activities appear normal in the CHO- cell ethyl methanesulfonate-sensitive mutant, EM9

M. La Belle 1, S. Linn 1 and L .H . T h o m p s o n 2

1Department of Biochemistry, University of California, Berkeley, California 94720 (U.S.A.) and 2Biomedical Sciences Division, L-452, Lawrence Livermore National Laboratory, P.O. Box 5507, Livermore, California 94550 (U.S.A.)

(Accepted 25 May 1984)

Summary

A study of the apurinic/apyrimidinic (AP) endonuclease activities of a mutant line of CHO cells, EM9, and its parental cell line, AA8, was undertaken to determine if the defective DNA repair exhibited by the mutant cell line after exposure to ethyl methanesulfonate was due to a defective AP endonuclease activity. Phosphocellulose chromatography of cell extracts resolved the AP endonuclease activities of both cell lines into two peaks as seen previously in mouse and human cells. No difference was found between the mutant and parental cell lines in the relative amount of AP endonuclease activity present in the two peaks.

Apurinic or apyrimidinic (AP) sites may arise in DNA due to the action of DNA glycosylases (Lin- dahl, 1979) or of DNA alkylating agents (Lawley and Brooks, 1963; Singer et al., 1978), or from spontaneous depurination which has been estimated to result in the loss of roughly 10000 purine bases from a mammalian cells genome per 24-h period (Lindahl and Nyberg, 1972). That such sites are potentially very detrimental to the cell is suggested by the presence of multiple pathways for repairing AP sites (Mosbaugh and Linn, 1980; Deutsch and Linn, 1979) and by studies demonstrating that AP sites inhibit DNA synthesis (Shearman and Loeb, 1979; Kunkel et al., 1981) and increase misincorporation and mutagenesis (Shearman and Loeb, 1979; Kunkel et al., 1981; Schaaper et al., 1983).

AP endonucleases hydrolyze phosphodiester bonds in DNA which are adjacent to AP sites. At present, virtually all purified AP endonucleases cleave the phosphodiester bond either 3' to the AP

site, producing 3'-deoxyribose and 5 ' -phoshomo- noester termini (a Class I AP endonuclease), or 5' to the AP site, producing 3 ' -hydroxyl nucleotide- and 5 '-phosphate termini (a Class II AP en- donuclease). [An exception is a human placental AP endonuclease which is reported to be capable of cleaving either 3' or 5' to the AP site (Grafstrom et al., 1982).] The 3'-deoxyribose ter- mini produced by Class I AP endonucleases must be removed either by an exonuclease or by the ac- tion of a Class II AP endonuclease before efficient DNA synthesis can occur (Mosbaugh and Linn, 1980). Previous work has shown that human and mouse cultured fibroblasts possess both Class I and Class II AP endonuclease activities which are resolvable by chromatography on phospho- cellulose (Mosbaugh and Linn, 1980; La Belle and Linn, 1984).

The CHO-ceU mutant strain, EM9, isolated as being hypersensitive to killing by ethyl methanesulfonate, shows cross-sensitivity to cer-

0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.

42

tain other simple alkylating agents and to ionizing radiation (Thompson et al., 1980b, 1982). EM9 cells are defective in restoring their DNA to high molecular weight after treatment with these agents. Moreover, they exhibit a highly elevated baseline

frequency of sister-chromatid exchanges (Thomp- son et al., 1982) which seem to result when DNA replication occurs on a template containing incor- porated 5-bromo-2 ' -deoxyuridine (Dillehay et al., 1983, 1984). Attempts to define the biochemical defect in EM9 have shown that DNA ligases (Chan et al., 1984) as well as the metabolism of po- ly(ADP-ribose) appear to be normal. In this study we examine two major AP endonuclease activities to determine whether an alteration has occurred in

either of them.

Materials and methods

Cells and culture conditions Mutant EM9 was isolated as described (Thomp-

son et al., 1980b) f rom the parental line, AA8 (Thompson et al., 1980a). Cells were grown at 37°C in c~-MEM with 10% fetal bovine serum (FBS, Sterile Systems, Inc.), penicillin G (50 units/ml), and streptomycin (50 #g/ml) in the presence of 5% CO2. Cells in 150 cmz T-flasks (Falcon) were subcultured while subconfluent. An- tibiotics and a - M E M were obtained f rom Gibco.

Preparation and depurination o f PM2 DNA Supercoiled [3H]DNA (9760 cpm/nmole , > 90%

Form I) was prepared f rom PM2 phage grown on a thymidine auxotroph, Bal 31-14, of Alteromonas espejiana, as described by Kuhnlein et al. (1976). PM2 [3H]DNA was depurinated to produce an average of 2.2 apurinic sites per circular PM2

genome by incubation for 15 min at 70°C in 10 mM sodium citrate (pH 5.0), 100 mM NaC1.

Fractionation of AP endonucleases 10 T-flasks of subconfluent cells were incubated

for 5 min at 37°C with 4 ml of 0.05% trypsin per flask, then the cells were pooled, mixed with 10 ml of FBS to inactivate the trypsin and washed with phosphate-buffered saline (PBS) by centrifugation

for 5 rain at 1000 rpm in a Beckman T J-6 tabletop centrifuge. The cells were then resuspended in 3 ml of 50 mM Tris-HCl (pH 7.5) and lysed by sonica- tion at 0°C for six, 10-sec intervals at 50 W using

a Biosonic sonicator equipped with a microprobe. The sonicate was examined microscopically to en- sure that complete disruption of the cells had oc- curred, then it was centrifuged at 12000 × g for 10 min at 4°C and the resulting pellet was discarded. The supernatant was brought to 0.4 M NaC1 and applied to a 0.8 cmZx 5 cm column of Whatman DE-52 cellulose pre-equilibrated with 50 mM Tris- HCI (pH 7.5), 0.4 M NaC1. The column was wash- ed with 20 ml of equilibration buffer and 1-ml fractions were collected. Fractions with significant A28o were pooled and dialyzed overnight at 4°C against two 0.5-#1 changes of 10 mM potassium phosphate (pH 7.5), 0.1 mM phenylmethylsulfonyl fluoride (PMSF). After dialysis, the extract was applied to a column of Whatman P11 phospho- cellulose equilibrated with I0 mM potassium phosphate (pH 7.5). The column was washed with 5 ml of equilibration buffer, then 3 ml of 50 mM potassium phosphate (pH 7.5). A 50-ml linear gradient from 50 mM to 350 mM potassium phosphate (pH 7.5) was then applied to the column to elute the remaining AP endonuclease activity. Fractions (1.5 ml) were collected and acetylated BSA was added to a final concentration of 1 mg/ml before assay for AP endonuclease activity.

Endonuclease assays Standard reaction mixtures contained 50 mM

Tris-HC1 (pH 7.5), 10 mM MgCI2, 0.02 mM depurinated or nondepurinated PM2 [3H]DNA, and 10 tzl of sample diluted 50-fold (column profile assays) or 100-fold (assays of pooled peak frac- tions) into 50 mM Tris-HCl (pH 7.5), 10 mM MgClz. For individual fractions 50-tzl reaction mix- tures were incubated at 37°C for 0 or 5 min; with pooled fractions duplicate 50-~1 assay mixtures were incubated for 0, 5, or 10 min, chilled on ice, and 200 #1 of 0 .01%.sodium dodecylsulfate, 0.25 mM EDTA (pH 7.0) was added to stop the reac- tion. The substrate DNA was then denatured by addition of 200 #1 of 0.3 M potassium phosphate

(pH 12.4) followed by a 2-min incubation at room temperature. Form I PM2 DNA was selectively renatured by addition of 100 #1 of 1 M KHzPO4 followed by 200/zl of 5 M NaC1. The samples were each diluted with 4 ml of 50 mM Tris-HC1 (pH 8.2), 1 M NaCl, then they were filtered through a Scheichner and Schuell, type BS85, nitrocellulose filter washed first with 5 ml of 50 mM Tris-HC1 (pH 8.2), 1 M NaC1, then with 5 ml of 0.3 M NaCl, 0.03 M sodium citrate. Filters were then dried and

¢0 ,,e

0

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E Q.

90-

80-

70-

60-

50-

40-

30-

20-

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O-

9° I 80

70

60-

50

40-

30-

20-

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AA8 ~ pp

; I . ~ 9

5 10 15 20 25 30 35 40

Fraction Number

E M 9

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S o •

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10 15 20 25 30 35 40

Fraction Number

I 360

320

280

240

200

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320 "i 2 8 0

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-120

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Fig. 1. Chromatography of AP endonuclease activities on phosphocellulose. AP endonuclease plus nonspecific en- donuclease activity (e) or nonspecific activity (zx) was deter- mined with partially depurinated or nondepurinated DNA substrate, respectively. Upper panel: Assay of pooled fractions indicated that 38°70 of the recovered activity was in the flowthrough and 62°70 in the retained fractions. Lower panel: Flowthrough and retained fractions contained 40°70 and 60% of the recovered activity, respectively.

43

counted by liquid scintillation. Since renatured Form I DNA is not bound by the filters under these conditions, the fraction of DNA bound to each filter can be used to calculate the mean number of nicks per PM2 genome by assuming a Poisson distribution of AP sites (Kuhnlein et al., 1976).

One unit of AP enonuclease activity produces 1 pmole of nicks specifically into AP DNA per min at 37°C.

Results and discussion

Upon phosphocellulose chromatography of ex- tracts f rom the repair-deficient EM9 cell line or the parental AA8 cell line, the AP endonuclease activi- ty was resolved into two major peaks (Fig. 1). The first peak of activity, AP endonuclease I, was not retained on the column under the conditions used and was recovered in the flowthrough, while the second peak, AP endonuclease II, eluted at about 240 mM potassium phosphate buffer. Rechro- matography of the f lowthrough activities on phosphocellulose showed no retention by the col- umn (data not shown), so that the flow-through position was not due to overloading of the resin. Thus CHO cells possess both retained and flowthrough AP endonuclease activities, as have been observed for both human (Mosbaugh and Linn, 1980) and mouse fibroblasts (La Belle and Linn, 1984). Assays of the pools of the two AP en- donuclease activities determined that for both the parental and repair-deficient cell lines, the AP en- donuclease I activity accounted for approximately 40°70 of the total activity recovered and the AP en- donuclease II activity comprised the remaining

60%. In conclusion, a comparison of the elution pro-

files of the AP endonuclease activities f rom the EM9 and AA8 cell lines demonstrated that the repair-deficient cells neither lack one of the two major AP endonuclease activities nor contain significantly different amounts of either of them. Since the DNA repair defect of EM9 cells would not appear to be due to an alteration of these AP endonuclease activities, a continued study of other components of DNA metabolism is needed.

44

Acknowledgements

T h i s w o r k was s u p p o r t e d b y N I H P o s t d o c t o r a l

F e l l o w s h i p G r a n t A G 0 5 2 4 6 to M . L a Bel le a n d b y

C o n t r a c t D E - A T 0 3 - 7 6 E V I 0 1 9 0 f r o m t h e D e p a r t -

m e n t o f E n e r g y , U . S . A .

References

Chan, J.Y.H., L.H. Thompson and F.F. Becker (1984) DNA ligase activities appear normal in the CHO mutant EM9, Mutation Res. (in press).

Deutsch, W.A., and S. Linn (1979) DNA binding activity from cultured human fibroblasts that is specific for partially depurinated DNA and that inserts purines into apurinic sites, Proc. Natl. Acad. Sci. (U.S.A.), 176, 141-144.

Dillehay, L.E., L.H. Thompson, J.L. Minker and A.V. Car- rano (1983) The relationship between sister-chromatid ex- change and perturbations in DNA replication in mutant EM9 and normal CHO cells, Mutation Res., 109, 283-296.

Dillehay, L.E., L.H. Thompson and A.V. Carrano (1984) DNA strand breaks associated with halogenated pyrimidine incor- poration, Mutation Res. (in press).

Grafstrom, R.H., N.L. Shaper and L. Grossman (1982) Human placental apurinic/apyrimidinic endonuclease, J. Biol. Chem., 257, 13459-13464.

Ikejima, M., D. Bohannon, D.M. Gill and L.H. Thompson (1984) Poly(ADP-ribose) metabolism appears normal in EM9, a mutagen-sensitive mutant of CHO cells, Mutation Res., 128, 213-220.

Kuhnlein, U., E.E. Penhoet and S. Linn (1976) An altered apurinic DNA endonuclease activity in group A and group D xeroderma pigmentosum fibroblasts, Proc. Natl. Acad. Sci. (U.S.A.), 73, 1169-1173.

Kunkel, T.A., C.W. Shearman and L.A. Loeb (1981) Mutagenesis in vitro by depurination of ~X 174 DNA, Nature (London), 291, 349-351.

La Belle, M., and S. Linn (1984) DNA repair in cultured mouse cells of increasing population doubling level, Mutation Res., 132.

Lawley, P.D., and P. Brooks (1963) Further studies on the alkylation of nucleic acids and their constituent nucleotides, Biocbem. J., 89, 127-138.

Lindahl, T. (1979) DNA glycosylases, endonucleases for apurinic/apyrimidinic sites, and base excision-repair, Progr. Nucleic Acid Res. Mol. Biol., 22, 135-192.

Lindahl, T., and B. Nyberg (1972) Heat-induced deamination of cytosine residues in deoxyribonucleic acid, Biochemistry, 11, 3610-3618.

Mosbaugh, D.W., and S. Linn (1980) Further characterization of human fibroblast apurinic/apyrimidinic DNA en- donucleases, J. Biol. Chem., 255, 11743-11752.

Schaaper, R.M., T.A. Kunkel and L.A. Loeb (1983) Infidelity of DNA synthesis associated with bypass of apurinic sites, Proc. Natl. Acad. Sci. (U.S.A.), 80, 487-491.

Shearman, C.W., and L.A. Loeb (1979) Effects of depurina- tion on the fidelity of DNA synthesis, J. Mol. Biol., 128, 197-218.

Singer, B., M. Kroger and M. Carrano (1978) 02- and O4-alkyl pyrimidine nucleosides: Stability of the glycosyl bond and of the alkyl group as a function of pH, Biochemistry, 17, 1246-1250.

Thompson, L.H., S. Fong and K. Brookman (1980a) Valida- tion of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured Chinese hamster ovary cells, Mutation Res., 74, 21-36.

Thompson, L.H., J.S. Rubin, J.E. Cleaver, G.F. Whitmore and K. Brookman (1980b) A screening method for isolating DNA repair-deficient mutants of CHO cells, Somatic Cell Genet., 6, 391-405.

Thompson, L.H., K.W. Brookman, L.E. Dillehay, A.V. Car~ rano, J.A. Mazrimas, C.L. Mooney and J.L. Minker (1982) A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister chromatid exchange, Mutation Res., 95, 427-440.