Apurinic/apyrimidinic-specific endonuclease activities from Dictyostelium discoideum
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304 Biochim.a et B.)phvs.'a A ~ta 824 ( 1 t;85) 304 312 Elsevier
Apur in ic /apyr imid in ic -speci f ic endonuclease activities from
Dictyostel ium discoideum
Robert B. Guyer and Reginald A. Deering *
Molecular and Cell Biology Program, The Pennsylvania State UniversiO,. 201 Althouse Laboratot T, Universi(v Park'. PA 16802 (U,S.A.)
(Received December 10th, 1984)
Key words: AP endonuclease; DNA repair; (D. discoideum)
Two apurinic/apyrimidinic- (AP-) specific endonuclease activities have been isolated from the cells of Dictyostelium discoideum by fractionation on DEAE-cellulose, CM-cellulose and Sephadex G-75. These activities, designated A and B, have apparent molecular weights of 49 000 and 40 000, respectively. Although their precise reaction optima differ somewhat, both A and B quantitatively nick AP DNA best at pH 8.0-8.5 in low salt (less than 100 mM NaCI); both require Mg 2+. These activities are apparently specific only for AP sites in DNA. The low activities observed on heavily ultraviolet-irradiated DNA, gamma-irradiated DNA and osmium tetroxide-treated DNA are consistent with the small numbers of secondary AP sites expected in these DNAs. Both A and B produce single-strand nicks in AP DNA that result in termini that serve as good primers for Escherichia coli polymerase I. Hence, A and B appear to be Class II AP endonucleases which yield 3'-OH termini at nicks on the 5' side of baseless sugars. It is unclear whether A and B are independently coded proteins, different post-translational modifications of the same gene product, or whether one is an artifact arising from the isolation. Many of the properties of these D. discoideum AP endonuclease activities are similar to those of the predominant AP endonucleases observed in bacterial, plant and animal cells. They will be of use in the characterization of excision repair in this organism.
DNA is altered in vivo by the inherent instabil- ity of certain bases, errors in replication and exog- enous damaging agents such as radiation and mutagenic chemicals. Excision repair is an im- portant mechanism for correcting these alter- ations. This is initiated by lesion-specific DNA glycosylases or endonucleases . Several glyco- sylases have been described that remove specifi- cally damaged bases from DNA, producing an AP site . This site can serve as the substrate for an
* To whom correspondence should be addressed. Abbreviations: AP, apurinic or apyrimidinic; Hepes, 4-(2-hy- droxyethyl)-i -piperazinethanesulfonic acid.
AP-specific endodeoxyribonuclease. Different AP endonucleases cleave in either of two ways, on the 3' side of the baseless sugar (Class I) or the 5' side (Class II) . The 3'-OH terminus arising from the latter activity serves as a good primer for DNA polymerase I, whereas that from the former does not . In some instances, the glycosylase and AP endonuclease activities are tightly associated, as for T4 endonuclease V and Micrococcus luteus ultraviolet damage-specific glycosylase/AP endo- nuclease [5-7]. Recently, Escherichia coli X-ray endonuclease (endonuclease III) and human lymphoblast endonuclease A have been shown to possess both glycosylase and AP endonuclease ac- tivities [8,9]. AP endonucleases with no associated glycosylase activity have been described for E. coli
0167-4781/85/$03.30 1985 Elsevier Science Publishers B.V. (Biomedical Division)
[10,11], M. luteus , Saccharomyces cerevisiae , Chlamydomonas , Phaseolus  and Drosophila . In mammals, AP endonucleases from rat liver , calf thymus , human skin fibroblasts , placenta [20,21] and HeLa cells  have been described. Reviews of damage- specific glycosylases and endonucleases are availa- ble [2,3,11].
The cellular slime mold, Dictyostelium dis- coideum, has been used as a model system for the study of repair in simple eukaryotes [23,24]. Re- pair-deficient mutants sensitive to various damag- ing agents have been isolated [25-27]. Following ultraviolet irradiation, thymine dimers are re- moved from the DNA in vivo [24,28]. Here we report the isolation and characterization of two AP endonuclease activities from this organism.
Materials and Methods
Materials Chemicals werc from the following sources:
growth media (Difco); dithiothreitol, Hepes, poly(ethylene glycol) (Mr approx. 8000), sodium lauroyl sarcosine, bovine serum albumin, pepsta- tin, leupeptin, ovalbumin, soybean trypsin inhibi- tor, dATP, dCTP and dGTP (Sigma); Dextran T-500 and Sephadex G-75 (10-40 /xm) (Pharmacia); DEAE-cellulose 32 and CM-cellulose 52 (Whatman); agarose (Standard, low Mr) and Protein Assay Dye Reagent (Bio-Rad); ethidium bromide (Calbiochem-Behring); osmium tetroxide, crystalline (Polysciences); bovine pancreatic DNAase I, crystalline, 2064 U/mg (Worthington); E. coli DNA polymerase I, endonuclease-free (Boehringer Mannheim); and [y-32p]dTTP (600 Ci/mmol) (New England Nuclear).
All buffers for columns and reactions were pre- pared in autoclaved containers using autoclaved, double-distilled H20 to minimize contamination by other nucleases. The pH values of all solutions were determined at room temperature. All steps in the enzyme purifications were performed at 0-4C. The proteinase inhibitors pepstatin (1/~g/ml) and leupeptin (5 #g/ml) were present at cell lysis and all stages of purification, storage and assay. These inhibitors and the maintenance of a nonacid pH may have provided some protection against the action of the proteinases of D. discoideum [29,30].
These inhibitors had no adverse effects on the endonuclease activities under investigation.
Preparation of the extract An axenic, repair-proficient strain of D. dis-
coideum, NP-2, was germinated from spores [23,25] and maintained in HL-5 medium . For enzyme isolations, 4 1 of cells were grown for about 60 h in a 7.5-1 New Brunswick Fermentor at 23C and harvested at mid-to-late log phase ((4-8).106 cells/ml) by centrifugation at 400 x g for 5 min. The 20-24 g cell pellet was washed three times by suspension in 250 ml phosphate-buffered saline (34 mM KH2PO4/16 mM Na2HPO4/9.9 mM NaC1/10 mM KC1, pH 6.5)  and centrifuga- tion. The washed cells were suspended at about 2.5.108 cells/ml in ice-cold, 50 mM Tris/5000 mM NaC1 (pH 8.0) , and sonicated for 5 x 60 s at 0C, using a Fisher Model 300 Sonic Dismem- brator with a 19-mm diameter tip. The super- natant (about 70 ml) from a 90-min centrifugation at 65 000 x g was adjusted to 6% (w/v) poly(ethyl- ene glycol) and 4% (w/v) Dextran T-500 in 40 mM Tris/4000 mM NaC1 (pH 8.0), followed by gentle stirring for 2 h at 4C and centrifugation at 20 000 x g for 20 min . The top (poly(ethylene glycol)) phase was saved; the lower (dextran) r" ase was reextracted with the top phase from a 'wash' mixture (35 ml of 40 mM Tris/4000 mM NaC1, pH 8.0), adjusted to 6% (w/v) poly(ethylene gly- col) and 4% (w/v) Dextran T-500, stirred 2 h and centrifuged. Following stirring for 1 h and centri- fugation, this second poly(ethylene glycol) phase was pooled with the first poly(ethylene glycol) extract phase [7,32]. These combined poly(ethylene glycol) phases were dialyzed overnight against two changes of 6 1 of 10 mM Tris-HC1 (pH 8.0)/0.1 mM dithiothreitol/1.0 mM EDTA/10% (v/v) eth- ylene glycol (buffer A) containing 5% (w/v) poly(ethylene glycol). A small amount of precipi- tate was removed by centrifugation and discarded.
D EAE-cellulose chromatography A column (2.5 x 33 cm) of DE-32 was equi-
librated in buffer A [7,18,32], and the dialyzed and centrifuged poly(ethylene glycol) extract super- natant was applied at 60 ml/h. The column was washed until the refractive index (due to poly(eth- ylene glycol)) returned to baseline and was devel-
oped with a 1200-ml linear gradient from 0-500 mM NaCl in buffer A. Column eluant was passed through an ISCO ultraviolet monitor (280 nm, 5 mm pathlength) and dripped into fraction collec- tor tubes containing pepstatin and leupeptin. The final concentrations of pepstatin and leupeptin in the 10-ml fractions were 1 and 5 ~g/ml, respec- tively. Pooled fractions were concentrated about 6-fold by ultrafiltration in a 50-ml, stirred cell containing a PM-10 membrane (Amicon).
CM-cellulose chromatography A column (1.5 23 cm) of CM-52 was equi-
librated in 10 mM NaH2PO4/10 mM CHBCOONa/0.1 mM dithiothreitol/1.0 mM EDTA/10% (v/v) ethylene glycol (pH 5.50) (buffer B). The 9-ml sample, adjusted to pH 5.5, dialyzed 2-4 h against buffer B, followed by centrifugation, was applied at 30 ml/h, and the column was developed with a 350-ml linear gradient, to 600 mM NaC1 in buffer B. Eluant was dripped into tubes containing pepstatin and leupeptin and enough 200 mM NazHPO 4 (adjusted to pH 8.0 with HC1) buffer to maintain sample pH at >/7.0. Pooled fractions were concentrated about 10-fold to 2 ml.
Sephadex G-75 A column (1.6 93 cm) of Sephadex G-75 was
equilibrated with 10 mM Hepes/500 mM NaC1/0.1 mM dithiothreitol/1.0 mM EDTA/10% (v/v) ethylene glycol (pH 8.0) (buffer C). A 1-ml sample from the CM-cellulose pooled concentrate was applied and eluted at 3.45 ml/h. The column was calibrated with bovine serum albumin, ovalbumin and soybean trypsin inhibitor.
Preparation of native and damaged PM2 DNA Covalently closed circular duplex PM2 DNA
was purified from a 20-1 culture as described by Espejo and Canelo  with recent modifications . This PM2 DNA (approx. 600 /xg/ml) was stored in 10 mM Tris/200 mM NaC1/2 mM EDTA (pH 8.3) at -75C.
AP DNA was prepared by heating PM2 DNA in 10 mM sodium citrate/100 mM NaC1 (pH 5.0) at 70C for 5 min/AP site per DNA molecule . The AP DNA was kept refrigerated at pH approx. 7.5 in the presence of 1 mM EDTA.
Ultraviolet-irradiated PM2 DNA was prepared at room temperature by irradiating 50-t~l aliquots with 254 nm light at a dose rate of 3 5 J /m 2 per s (Model J225 dosimeter, UltraViolet Products) . The ultraviolet-irradiated DNA (150 t~g/ml) in 3 mM Tris/55 mM NaCI/1 mM EDTA (pH ap- prox. 7.5) was stored at 4C. The number of pyrimidine dimers in ultraviolet-irradiated PM2 DNA was calculated from the published data of Woodworth-Gutai et al. .
Gamma-irradiated PM2 DNA was prepared by irradiation of DNA with 6Co gamma rays at 0C in 10 mM Tris/1 mM EDTA/50 mM KI (pH 8.0) . The dose rate in the Gammacell 200 (Atomic Energy of Canada) was 8.2 krad/min. The 50 t~g/ml solution of gamma-irradiated DNA was dialyzed against 10 mM Hepes/50 mM NaCI/5 mM MgC12 (pH 8.0) (buffer D).
Osmium tetroxide-treated PM2 DNA was pre- pared by the method of Armel et al. . Briefly, PM2 DNA (50 ~g/ml) was dialyzed extensively against 0.12 mM sodium cacodylate (pH 7.0). The DNA was heated to 65C for 10 rain as was an equal volume of freshly diluted 0.2% osmium tetroxide in the same buffer. Then the osmium tetroxide was added to the DNA and the mixture was incubated for 15 rain at 65C. Four extrac- tions (of osmium tetroxide) with ice-cold diethyl ether were followed by exhaustive dialysis of the aqueous phase against 10 mM Tris/1 mM EDTA (pH 8.0).
Endonuclease nicking assay Unless otherwise noted, the conditions listed in
this section apply to all assays (i.e., standard as- say) in this report. Buffer D (see above) was the usual assay buffer. The DNA storage solution contributed 7 mM NaC1 to the reactions. PM2 DNA and damaged DNAs were diluted to 20 /~g/ml in buffer D containing 100/~g bovine serum albumin/ml. The bovine serum albumin was pre- viously heat-treated at 100C for 5 rain at pH 3.0 to minimize the activity of any contaminating nucleases . The substrate for the standard AP endonuclease assays was PM2 DNA containing six AP sites/molecule. Enzyme samples were diluted in buffer D containing 1 /~g pepstatin/ml, 5 /~g leupeptin/ml, 500 t~g heat-treated bovine serum albumin/ml and 10% (v/v) ethylene glycol (buffer
E). The reaction mixture contained 30 #1 buffer D, 20/tl (0.40 #g) DNA and 20 ~tl enzyme. After 60 rain at 30C, the reaction was stopped by adding 20 Itl of 10% (w/v) sodium lauroyl sarcosine/30% (w/v) sucrose/0.03% bromophenol blue.
A 45-/~1 aliquot of the stopped reaction mixture was applied to wells of a horizontal 1% agarose gel (20 25 cm; Bethesda Research Laboratories). The unnicked DNA (Form I) was separated from the nicked species (Form II, open circular or Form III, linear) by electrophoresis at 50 V for 12-18 h [40,41]. The electrophoresis buffer (50 mM Tris/20 mM sodium acetate/18 mM NaCI/2 mM EDTA (adjusted to pH 8.0 with acetic acid)  was recycled between buffer chambers at approx. 1 1/h. Gels were stained for 45 min in the dark in 1100 ml of electrophoresis buffer containing 1 /~g ethidium bromide/ml, illuminated with 302 nm light (Fotodyne), and photographed through glass and Wratten 2B and 29 filters [41,43]. The Kodak Tri-X film was developed according to the manu- facturer's instructions, and the density of entire bands was measured with a Quick Scan Jr TLC unit (Helena). The densities were linear for 0-0.20 #g DNA/band. The nicking activity (average number of single-strand breaks/DNA molecule) was calculated, assuming the Poisson distribution of nicks, as - In of the proportion of superhelical molecules remaining unnicked . The product of the nicking of Form I DNA was predominantly Form II. However, a small amount of Form III DNA was observed with reactions that produced more than approx. 1.5 nicks/DNA molecule. One unit of activity was defined as the amount of enzyme that hydrolyzed one femtomole of phos- phodiester bonds per minute under the standard assay conditions described above.
Protein assay The protein concentration of enzyme samples
was determined by the method of Bradford  using ovalbumin as the standard.
DNA polymerase assay The determination of whether nicks were pro-
duced on the 3' or 5' side of the AP site was performed as described by Mosbaugh and Linn . The PM2 DNA had an average of 1.5 AP sites/molecule. Bovine pancreatic DNAase I was used as a positive control .
Purification of the enzyme(s) The elution profile of a typical DEAE-cellulose
column is shown in Fig. 1. Two peaks of AP endonuclease activity, designated 'A (fractions 20-25) and B (fractions 31-36), eluted early in the gradient along with considerable amounts of other proteins. Since these fractions were assayed in 57 mM NaC1, activity B may have been under- estimated by about 50% (see Fig. 5). Also, two peaks of activity that nicked undamaged PM2 DNA at pH 7-9 eluted in fractions 13-19 and 24-33 (not shown). Most of these nonspecific ac- tivities eluted before peak A and between peaks A and B. In addition, a major nonspecific endo- nuclease activity that was active at acid pH eluted in fractions 56-64 (Guyer, R.B., Skantar, A.M. and Deering, R.A., unpublished data).
Peaks A and B from the DEAE-cellulose col- umn were concentrated and fractionated on CM- cellulose. Fig. 2 shows a composite of column...