Volume 5 Number 9

September 1978

Nucleic Acids Research

Apurinic endonucleases from Saccharomyces cerevisiae

Paul R. Armel and Susan S.Wallace

Department of Microbiology, New York Medical College, Valhalla, NY 10595, USA

Received 26 June 1978

ABSTRACT Three endonuclease activities have been partially purified from Saccharomyces cerevisiae on the basis of their activity against x--irradiated closed-circular supercoiled bacteriophage PM2 DNA. These endonucleases also nick apurinic DNA and two out of the three activities incise DNA UV-irradiated with high doses. The endonuclease activities have also been distinguished on the basis of their magnesium requirement and sensitivity to EDTA.

INTRODUCTION Endonucleases which incise apurinic DNA have been purified from a variety of sources including Escherichia coli,1' 2 calf thymus, 3 rat liver,4 plant embryos5 (Phaseolus multiflorus), human placental tissue 6 and cultured human lymphoblasts. 7 It is assumed that these.apurinic endonucleases are involved in the first steps of an excision repair process in which the substrate apurinic sites have been formed either directly by a physical or a chemical agent such as x-rays or alkylating agents, or indirectly by the removal of a damaged or incorrect base residue by an N-glycosylase. The latter class of enzymes has been shown to function on uracil-containing DNA8 '9 as well as DNA containing 06-methylguanine, 10 3-methyladenine, 10,11 and 7-bromomethyl-12-methylbenz(a)anthracene. 12 Verification of the role of apurinic enzymes in an excision repair process awaits the detailed study of mutants deficient in these enzymes. It has been established that certain E. coli mutants which appear to have mutations in a gene coding for an apurinic endonuclease are also sensitive to methyl methanesulfonate, an alkylating agent which introduces apurinic sites into DNA. 13, 14, 15 The present study describes the partial purification from Saccharomyces cerevisiae of three endonuclease activities which incise DNA containing apurinic sites.

C) Information Retrieval Limited 1 Falconberg Coun London Wl V 5FG England


Nucleic Acids Research MATERIALS AND METHODS Yeast and bacterial strains. Bal 31-14 (thymine requiring auxotroph) and PM2 bacteriophage were obtained from H.B. Grey, Jr. Saccharonyces cerevisiae haploid strain LP98-1D was obtained from Louise Prakash. Yeast were routinely grown in YPD medium containing 1% yeast extract, 2% peptone and 2% dextrose. Chemicals. All chemicals were of analytical grade unless otherwise specified. Tris (enzyme grade) and ammonium sulfate (special enzyme grade) were obtained from Schwarz Mann. 3H-thymidine was purchased from ICN. DNA preparation. 3H PM2 was prepared as previously described.'6 DNA concentrations for different preparations ranged from about 8 to 10 pg per ml at a specific activity of approximately 50,000 cpm/pg. Preparation of substrates. X-irradiated PM2 DNA substrate was prepared by irradiating small volumes (33-100 il) of 3H PM2 DNA in 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM potassium iodide (KI) in air at room temperature with a Picker x-ray source run at 60 kVp and 2.5 mA. The dose rate was 27.6 Krad/min as determined by using T4 bacteriophage as a dosimeter. Apurinic PM2 DNA was prepared by the method of Lindahl.17UV irradiation was accomplished by using a General Electric germicidal lamp powered through a Solar constant voltage regulator giving a dose rate of approximately 1.0 joule/meter2/sec as determined by a Blak-ray ultraviolet dosimeter. Endonuclease reactions. Samples of unirradiated, x-irradiated, apurinic, or UV-irradiated 3H PM2 DNA were mixed with or without yeast protein fractions in 100 pil reaction mixtures containing 50 mM potassium phosphate pH 7.5, 1 mM mercaptoethanol and 5 pg of calf thymus DNA. Reaction mixtures contained either 2 mM ATP and 8 mM MgCl2 or 8 mM EDTA. Reactions were incubated at 300C for 30 minutes and terminated by the addition of 33 PI of 4 M KCl and quick freezing in a dry ice-acetone bath. For sucrose gradient sedimentation, 8 ul of 200 mM EDTA was added to the thawed reaction mixtures. Sucrose gradient centrifugation. Samples to be analyzed by neutral sucrose sedimentation were layered on 5-20% gradients containing 10 MM TrisBase pH 7.5, 1 M KCl and 1 mM EDTA. 5-20% alkaline sucrose gradients contained 0.9 M NaCl and 0.15 N NaOH. Neutral gradients were centrifuged at 45,000 rpm at 40C in a Beckman SW 50.1 rotor for 200 minutes while alkaline gradients were centrifuged in the same rotor at 4°Cfor 100 minutes. Gradients were collected in 33 equal fractions in dram glass vials. After addition of 3.5 ml of Liquiscint (National Diagnostics) for neutral gradients or 3.5 ml Liouiiscint and 0.2 ml distilled H20 for alkaline gradients, the 3348

Nucleic Acids Research dram vials


placed in standard glass scintillation vials and monitored

for 3H CPM using a Searle scintillation counter. Total alkali-labile lesions plus strand breaks were determined by incubation of the reaction mixture for 16 hours at room temperature with an equal volume of 1.0 M glycine, pH 13 followed by analysis on alkaline sucrose gradients. In all sedimentation analyses, remaining Type I PM2 DNA was normalized to an untreated control. Strand breaks were calculated assuming a Poisson distribution. Other methods. DNA agarose was prepared by the method of Shaller et al 18 except that 4% w/v agarose was used instead of 8%. Protein determinations were made using the Biorad Protein Assay (Biorad Laboratories).

RES ULTS Enzyme purification. Yeast cells were grown with aeration in YPD medium to a concentration of approximately 8 x 107/ml. The cells were then chilled to 0°C after which they were harvested by centrifuging at 10,000 g for 15 minutes. All subsequent purification steps were carried out at 40C. The collected cell pellet was placed in a volume of 50 mM KPO4 pH 8.0, 1 mM E DTA, 1 mM HSETOH, 2.5 M NaCl, 1 mM phenylmethylsulfonylfluoride, 10% glycerol (Buffer A) equal to the volume of the cell mass. This mixture was then homogenized with cooling in a Braun Homogenizer MSK for 150 seconds. The homogenate was spun at 10,000 g for 10 minutes. The supernatant (Fraction I) was brought to a pH of approximately 7 by the addition of a small amount of saturated Tris-Base. Neutralized Fraction I was centrifuged at 140,000 g for one hour. The supernatant (Fraction II) was collected with a Pasteur pipette taking care to avoid the membranes. To remove nucleic acids, Fraction II was brought to 1.5 times its original volume by the addition of 50 mM KPO4 pH 8.0, 1 mM EDTA, 1 mM HSETOH, 1 M NaCl, 30% polyethylene glycol 6,000, 10% glycerol (Buffer B). This solution was stirred for 30 minutes and subsequently centrifuged for 30 minutes at 1Z,000 g. The supernatant (Fraction III) was extensively dialyzed against 20 mM KPO4 pH 8.0, 1 mM EDTA, 1 mM HSETOH, 0.1 M KCl, 10% glycerol (Buffer C). Dialyzed Fraction III contained a precipitate which was removed by centrifugation at 10,000 g for 10 minutes. The supernatant was loaded onto a DNA agarose column previously equilibrated with Buffer C. The enzyme activities were eluted stepwise with 20 mM KPO4 pH 8.0, 1 mM EDTA, I{SETOH, 10% glycerol, containing 0.2 M KCl, 0.3 M KCl, 0.4 M KCl, 0.5 M KCl, and 1.0 M KCl respectively. Two fractions of one column volume each were collected at 3349

Nucleic Acids Research each salt concentration. The column flow rate was approximately two column volumes per hour. The fractions were concentrated by dialysis against Buffer D (Buffer C minus KCl) made 30% w/y with respect to polyethylene glycol 20,000. The concentrated fractions were dialyzed further against Buffer D after which they were assayed. Assays were performed on column eluates using x-irradiated (20 krads) PM2 DNA substrate. The results from one such chromatograph are shown in Figure 1. Under these conditions, three peaks of endonuclease activity against x-irradiated DNA substrate were routinely observed. Fractions IV A,






0.8 _








0.2 0,



4 6 8 Fraction Number


Fig. 1 DNA agarose chromatography of endonuclease activity (solid line). KCI gradient

(dashed line). IV B, and IV C elute at approximately 0.2 M, 0.35 M, and 0.6 M KCl respectively. If one assumes that these activities are present in equal amounts in crude cell extracts, Fractions IV A, IV B and IV C exhibit approximately 3, 20 and 75 fold purifications respectively. In another preparation Fraction IV C was purified about 290 fold using this procedure. Substrate specificity. Figure 2 shows the x-ray survival of PM2 Type I DNA measured under neutral conditions to determine strand breaks; measured immediately in alkali to determine fast converting alkali-labile lesions plus strand breaks; and incubated at high pH for 16 hours to measure total alkalilabile lesions plus strand breaks.19'20 When the x-irradiated DNA was incubated with saturating amounts of Fraction IV C and analyzed on neutral gradients, conversion of Type I to Type II was equivalent to about 70% of the total alkali-labile lesions. Thus, it appeared from quantitative


Nucleic Acids Research z

100 80


60 cL 06

N ._



.5 E 0

Dose (kilorods) Fig. 2 Analysis of strand breaks produced

in PM2 DNA.

X-ray induced single strand

breaks -fast-converting alkali labile lesions 0 X-ray induced single strand breaks + endonuclease sensitive sites * total alkali labile lesions. considerations that Fraction IV C was acting at apurinic sites. Figure 3A and B shows the results of a reaction using apurinic DNA and an aliquot of Fraction IV C. Figure 3C illustrates the conversion into strand breaks of apurinic sites in PM2 DNA by incubation at high pH and subsequent analysis on alkaline sucrose gradients. This apurinic DNA preparation contained 1.97 apurinic sites per PM2 genome. The expected value was 2 sites per PM2 genome based on an 8 minute incubation in acid medium at 700C as determined by Lindahl and Andersson. 17 It can be seen that Fraction IV C converts 64% of

12 0



c 8 10 0


4 2










Fraction Number Fig. 3 Sucrose gradient analysis of apurinic PM2 DNA.A: Neutral sucrose gradient profile of apurinic PM2 DNA. B: Neutral sucrose gradient profile of apurinic PM2 DNA + endonuclease fraction IV C. C Alkaline sucrose gradierit profile of apurinic PM2 DNA after 16 hours incubation at high pH. 3351

Nucleic Acids Research the potential alkali-labile DNA lesions to breaks. In other experiments, under saturating conditions, this conversion was quantitative. The three peak fractions were then tested on DNA containing 1.5 or 1.75 apurinic sites per PM2 genome and their Mg++ and ATP requi'rements ascertained. The results under conditions where Mg++ and ATP are present or absent, or when EDTA is present, are shown in Table I. It can be seen that in the presence of Mg++ and ATP, all three Fractions are maximally active. When Mg++ and ATP are absent from the reaction mix, Fractions IV A, IV B, and IV C show reduced activity on apurinic DNA. However, Fractions IV A and IV B show a greater dependence upon Mg++ and ATP than Fraction IV C. The latter appears to be stimulated by Mg++ and ATP. Further, the activities of Fractions IV A and IV B are insensitive to EDTA in the absence of Mg++ and ATP while that of IV C shows a significant additional reduction under these conditions. Tabl e I

Effect of Yeast Fraction IV endonuclease activities on apurinic PM2 DNA

Breaks IV A +



(8mM Mg ++ 2m" ATP) (8nM Mg+++ 2mM ATP) (8mM Mg ++ 2uK ATP) +

/ molecule

/ apurinic site*


IV C 0.75










*Reactions were performed under non-saturating conditions.

Fractions IV A, B and C were then tested on UV-irradiated DNA substrates. The results are shown in Table II. At doses (150 J/m2) giving approximately 16 thymine dimers per PM2 genome 7 almost no activity can be seen in any of the three fractions. However, at doses (900 J/m2) giving about 95 thymine dimers per PM2 gemone, little activity was observed with Fraction IV A, but Fractions IV B and C produced about 0.45 nicks/molecule. Under the conditions used for the above reactions (presence of competing unirradiated calf thymus DNA), little or on endonuclease activity was seen on unirradiated PM2 DNA. Since activity against unirradiated DNA was found in only this particular preparation, no compensation for this activity of Fractiori IV A was made in the data presented here. It should be noted, however, that nonspecific breakdown of DNA does occur if calf thymus DNA is not included in the reaction mixture. The results obtained with unirradiated, x-irradiated, and UV-irradiated DNA substrates are sunmmarized in Table II. 3352

Nucleic Acids Research Table I I Substrate specificity of Fraction IV endonuclease activities from yeast

Enzye Breaks/PM2 Unirradiated DNA


Molecule/30 minutes

Per Kilorad UV-irradiated DNA UV-irradiated DNA X-irradiated 16 thymine dimers/ 95 thymine dimers/ DfA molecule molecule














0.20 0.45 0.47

a. nicking of unirradiated DNA was not observed in other preparations under the reaction conditions used. Enzyme reactions were carried out as close to saturation (as determined with x-irradiated DNA) as possible.

DISCUSSION Chlebowicz and Jachymczyk21have reported that labeled nucleotides are excised from alkylated E. coli DNA by crude extracts of S. cerevisiae. The inference is that apurinic endonuclease activity was required prior to exonuclease mediated excision. Pyrimidine dimers have also been shown to be excised from UV-irradiated S. cerevisiae.22' 23, 24 However, the examination of repair in yeast has generally depended on genetic studies (see Cox and Game 25 for a discussion of this approach). These studies have yielded a wealth of information concerning the repair pathways in yeast of which there appear to be at least three (other than photoreactivation) for ultraviolet irradiated DNA. 26 One pathway seems to be a classic excision pathway consisting of a number of genetic loci. 27 The nature of the other two UV repair pathways is less well established. One of these appears to be a recombinational repair pathway, 28, 29 while the other is an error prone system. These latter two are the only pathways thus far implicated in repair of x-ray induced damages. 28, 29 However, the presence of apurinic endonucleasesin yeast implicates a form of excision repair process for x-ray and chemically induced damages which leads to the formation of apurinic sites. Methyl methanesulfonate sensitive mutants defective in repair 30 have been isolated, and these will be examined for the presence or absence of the above described apurinic endonuclease activities. Several nuclease activities of S. cerevisiae have been isolated.31'32'33 Two of these activities, endonuclease A 31 and endonuclease °C 33 act preferentially on single-stranded DNA in the presence of either Mg++ or Mn++. Endonuclease B 32 makes single stranded scissions in double-stranded DNA when Mn++ is present and is inactive in the presence of Mg++. Endonuclease C32 3353

Nucleic Acids Research makes Single-stranded scissions in double-stranded DNA in the presence of Mg++, and double-stranded scissions in the presence of Mn++. In addition to the above, at least one DNase activity has been isolated from the mitochondria of S. cerevisiae.34 This nuclease degrades double-stranded DNA at neutral pH in the presence of Mg++ and is stimulated by ethidium bromide and other DNA intercalating agents. The presence of three peaks of x-ray specific endonuclease activity certainly does not establish the existence of three distinct enzymes. These may represent

three forms of the

same enzyme.

However, the fact that Fraction

IV A is almost conpletely inactivated in the absence of Mg++ and&ATP and has little activity against UV-irradiated DNA while IV C is active in the absence of Mg++ and ATP and nicks DNA irradiated by high doses of ultraviolet light indicates that there are at least two different apurinic endonucleases present in S. cerevisiae. In keeping with the terminology initiated by Pifnon31, 32 we suggest that Fractions IV A and IV C apurinic activities be called endonucleases D and E respectively. The activity of Fraction IV B has not been named since the differentiation of this activity on the basis of Mg++ and ATP requirement and specificity for UV-irradiated DNA from those of IV A and IV C is not clear. It is interesting that Fractions IV B and C have activity against DNA irradiated with high doses of ultraviolet light. The extent of conversion of Type I to Type II PM2 DNA cannot be accounted for by reaction specific for pyrimidine dimers or for apurinic sites. The latter are produced at this ultraviolet dose only to the extent of about 0.07 apurinic sites per PM2 molecule. It is more likely that another kind of photo product is being recognized, possibly those of the 5,6 dihydroxy-dihydrothymine type. However, if the latter are recognized in UV-irradiated DNA, one would expect incision at such alkali-stable sites which have been shown to occur in x-irradiated DNA.19%20 However, the failure to produce a greater number of breaks in x-irradiated DNA by enzyme treatment than is achieved by incubation in alkali does not a priori mean that only apurinic sites in this substrate are recognized by the endonucleases described here. A portion of the alkali-labile bonds and a portion of other alkali-stable sites may in fact be incised. From the data presented here, it is not possible to distinguish among these possibilities. Thus, the apurinic endonuclease fractions tested might contain more than one endonuclease with different specificities or a single endonuclease with more than one activity. The latter case has several precedents in E. coli. 35'36'37


Nucleic Acids Research Although the exact in vivo functions of the nucleases previously reported from S. cerevisiae remain unclear, it appears that these enzymes are not primarily concerned with DNA repair since they were purified on the basis of their activity against undamaged DNA. The endonuclease activities in Fractions IV A, IV B, and IV C are likely candidates for repair enzymes since they act preferentially on irradiated or depurinated DNA. Further purification of endonucleases D and E is currently in progress.

Acknowledgement This research was supported by grant number CA 21342 awarded by the National Cancer Institute, US DHEW. References

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12.

13. 14. 15. 16.

17. 18. 19.

20. 21. 22.

23. 24. 25. 26.

Verly, W.G., and Rassart, E. (1975). J. Biol. Chem. 250, 8214-8219. Ljungquist, S. (1977). J. Biol. Chem. 252, 2808-2814. Ljungquist, S., and Lindahl, T. (1974). J. Biol. Chem. 249, 1530-1535. Verly, W.G., and Paquette, Y. (1973). Can. J. Biochem. 51, 1003-1009. Thibodeau, L., and Verly, W.G. (1977). J. Biol. Chem. 252, 3304-3309. Linsley, W.S., Penhoet, E.E., and Linn, S. (1977). J. Biol. Chem. 252, 1235-1242. Brent, T.P. (1976). Biochim. Biophys. Acta 454, 172-183. Lindahl, T. (1974). Proc. Nat. Acad. Sci. USA 71, 3649-3653. Cone, R., Duncan, J., Hamilton, L., and Friedberg, E.C. (1977). Biochemistry 16, 3194-3201. Kirtikar, D.M., and Goldthwait, D.A. (1974). Proc. Nat. Acad. Sci. USA 71, 2022-2026. Laval, J. (1977). Nature 269, 829-832. Kirtikar, D.M., Dipple, A., and Goldthwait, D.A. (1975). Biochemistry 14, 5548-5553. Yajko, D.M., and Weiss, B. (1975). Proc. Nat. Acad. Sci. USA 72, 688-692. Kirtikar, D.M., Cathcart, G.R., White, J.G., Ukstins, I., and Goldthwait, D.A. (1977). Biochemistry 16, 5625-5630. Ljungquist, S., Lindahl, T., and Howard-Flanders, P. (1976). J. Bact. 126, 646-653. Strniste, G.F., and Wallace, S.S. (1975). Proc. Nat. Acad. Sci. USA 72, 1997-2001. Lindahl, T., and Andersson, A. (1972). Biochemistry 11, 3618-3623. Schaller, H., Nu'sslein, C., Bonhoeffer, F.J., Kurz, C., and Nietzschmann, I. (1972). Eur. J. Biochem. 26, 474-481. Armel, P.R., Strniste, G.F., and Wallace, S.S. (1977). Radiat. Res. 69, 328-338. Katcher, H.L., and Wallace, S.S. (1978). Int. J. Rad. Biol. in press. Chlebowicz, E., and Jachymczyk, W.J. (1977). Molec. Gen. Genet. 154, 221-223. Unrau, P., Wheatcroft, R., and Cox, B.S. (1971). Molec. Gen. Genet. 354, 359-362. Ferguson, L.R., and Cox, B.S. (1974). Molec. Gen. Genet. 135, 87-90. Prakash, L. (1975). J. Mol. Biol. 98, 781-795. Cox, B. and Game, J. (1974). Mutation Res. 26, 257-264. Game, J.C. and Cox, B.S. (1973). Mutation Res. 20, 35-44. 3355

Nucleic Acids Research 27. Haynes, R.H. (1975). in Molecular Mechanisms for Repair of DNA part B eds. Hanawalt, P.C. and Setlow, R.B. (Plenum Press, New York and London) 529-540. 28. Lawrence, C.W., and Christensen, R. (1976). Genetics 82, 207-232. 29. Resnick, M.A., and Martin, P. (1976). Molec. Gen. Genet. 143, 119-129. 30. Prakash, S., and Prakash, L. (1977). Genetics 87, 229-236. 31. Piinion, R. (1970). Biochemistry 9, 2839-2845. 32. Pinon, R., and Leney, E. (1975). Nucleic Acids Res. 2, 1023-1042. 33. Bryant, D.W., and Haynes, R.H. (1978). Can. J. Biochem. 56, 181-189. 34. Paoletti, C., Couder, H., and Guerineau, M. (1972). Biochem. Biophys. Res. Commun. 48, 950-958. 35. Radman, M. (1976). J. Biol. Chem. 251, 1438-1445. 36. Gates, III, F.T., and Linn, S. (19m77. J. Biol. Chem. 252, 2802-2807. 37. Gates, III, F.T., and Linn, S. (1977). J. Biol. Chem. 252, 1647-1653.


Apurinic endonucleases from Saccharomyces cerevisiae.

Volume 5 Number 9 September 1978 Nucleic Acids Research Apurinic endonucleases from Saccharomyces cerevisiae Paul R. Armel and Susan S.Wallace De...
761KB Sizes 0 Downloads 0 Views