EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 35, No. 2, 106-112, April 2003 Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli Yun-Song Lee1,3 and Myung-Hee Chung2 Introduction 1Division of Pharmacology 8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a muta- Department of Molecular and Cellular Biology genic adduct formed by reactive oxygen species Sungkyunkwan University School of Medicine (Kasai and Nishimura, 1984). As a structural prefe- Suwon 440-746, Korea rence, adenine is frequently incorporated into oppo- 2Department of Pharmacology site template 8-oxo-G (Shibutani et al., 1991), and 8- Seoul National University College of Medicine oxo-dGTP is incorporated into opposite template dA Jongno-gu, Seoul 110-799, Korea during DNA synthesis (Cheng et al., 1992). Thus, un- 3Corresponding author: Tel, 82-31-299-6190; repaired, these mismatches lead to GT and AC trans- Fax, 82-31-299-6209; E-mail, [email protected] versions, respectively (Grollman and Morya, 1993). In Escherichia coli, several DNA repair enzymes, Accepted 29 March 2003 preventing mutagenesis by 8-oxo-G, are known as the GO system (Michaels et al., 1992). The GO system Abbreviations: 8-oxo-G, 8-oxo-7,8-dihydroguanine; Fapy, 2,6-dihy- consists of MutT (8-oxo-dGTPase), MutM (2,6-dihydro- droxy-5N-formamidopyrimidine; FPG, Fapy-DNA glycosylase; BER, xy-5N-formamidopyrimidine (Fapy)-DNA glycosylase, base excision repair; AP, apurinic/apyrimidinic; dRPase, deoxyribo- Fpg) and MutY (adenine-DNA glycosylase). 8-oxo- phosphatase GTPase prevents incorporation of 8-oxo-dGTP into DNA by degrading 8-oxo-dGTP. Adenine-DNA glyco- sylase preferentially excises A paired with 8-oxo-G, restoring the 8-oxo-G:C pair, a substrate for Fpg. Abstract Fpg has multiple enzymatic activities. Through its glycosylase activity, it removes Fapy and 8-oxo-G from 8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a oxidatively damaged DNA (Tchou et al., 1991), and mutagenic adduct formed by reactive oxygen it has apurinic/apyrimidinic (AP) lyase activity to remove species. In Escherichia coli, 2,6-dihydroxy-5N-for- the AP site by successive β- and δ-elimination mecha- mamidopyrimidine (Fapy)-DNA glycosylase (Fpg) re- nisms, which results in a single nucleotide gap with moves this mutagenic adduct from DNA. In this 3’ and 5’ phosphate termini at the gap (Tchou et al., report, we demonstrate base excision repair (BER) 1991; Bhagwat and Gerlt, 1996). In addition, it has synthesis of DNA containing 8-oxo-G with Fpg in 5’ deoxyribophosphatase (dRPase) activity to remove vitro. Fpg cut the oligonucleotide at the site of 5’ deoxyribophosphate from DNA strands (Graves et 8-oxo-G, producing one nucleotide gap with 3’ and al., 1992). 5’ phosphate termini. Next, 3’ phosphatase(s) in Various kinds of DNA glycosylases specific to each the supernatant obtained by precipitating a crude of the modified bases have been known to initiate extract of E. coli with 40% ammonium sulfate, base excision repair (BER) DNA synthesis by remov- removed the 3’ phosphate group at the gap, thus ing the modified DNA bases. In the case of the DNA exposing the 3’ hydroxyl group to prime DNA syn- glycosylases with AP lyase activity to induce β- and thesis. DNA polymerase and DNA ligase then com- δ-elimination, short patch BER synthesis has been pleted the repair. These results indicate the biolo- proposed (Memisoglu and Samson, 2000). Initially, gical significance of the glycosylase and apurinic/ the glycosylases produce one nucleotide gap with 3’ apyrimidinic (AP) lyase activities of Fpg, in concert and 5’ phosphate termini at the gap through gly- cosylase and AP lyase activities. Then, 3’ phospha- with 3’ phosphatase(s) to create an appropriately tase removes the 3’ terminal phosphate at the gap, gapped substrate for efficient BER synthesis of providing a 3’ prime end for the insertion of a single DNA containing 8-oxo-G. nucleotide. Thereafter, DNA polymerase inserts one nucleotide and DNA ligase seals the nick to complete Keywords: DNA repair; Escherichia coli; mutagenesis; short patch BER DNA synthesis. Here, we demon- oxiative stress; reactive oxygen species; phosphatases strate in vitro short patch BER synthesis of DNA containing 8-oxo-G by Fpg based on this proposed repair model. Base excision repair of DNA containing 8-oxoguanine 107 Material and Methods ATCAGTGoxoCACCATCCCGGGTCGTTTTACAACGT- CGTGACT 3') was labeled at the 3' end with a 3' Materials end-labeling kit and [α-32P]cordycepin ATP, and an- Exonuclease III-deficient E. coli (BW 9053) was used nealed to its complementary oligonucleotide as des- for the purification of Fpg and the AS 40/100 fraction. cribed previously (Chung et al., 1991). For identi- Purification procedures were performed using HPLCs fication of the 3' or 5' termini at the incision site by (Model 302 pump, Model 811B gradient mixer, Model Fpg or the AS 40/100 fraction, single-stranded 5' end- 115 UV detector; Gilson, France), and a conventional labeled oligonucleotide (21-mer) or single-stranded 3' end-labeled oligonucleotide (46-mer) was incubated gel filtration chromatography (2.6×100 cm column o packed with Sephacryl S-200; Amersham Pharmacia, with piperidine at 90 C for 30 min (Chung et al., UK). Columns for phenyl HPLC (Phenyl-5PW, 21.5× 1992). To confirm removal of 3' terminal phosphate, 150 mm), DEAE HPLC (DEAE-5PW, 21.5×150 mm), synthetic 12-mer oligonucleotide without 3' terminal heparin affinity HPLC (Heparin-5PW, 7.5×75 mm) phosphate was labeled at 5' end. and hydroxylapatite HPLC (HA 1000, 7.5×75 mm) were from Toyo Soda (Japan). Radioactive nucleoti- Enzyme reactions des were obtained from Amersham Pharmacia (UK) Formation of a single nucleotide gap by Fpg or NEN (Boston, MA). T4 polynucleotide kinase and 3' end-labeling kits and nucleotides were from Roche Fpg or the AS 40/100 fraction was incubated with (Germany). T4 DNA polymerase, T4 DNA ligase and 0.4 pmol of 5' end-labeled or 3' end-labeled substrate gene 32 product were from BioRad (Hercules, CA). DNA in 50 µl of a reaction mixture containing 50 µM All other chemicals were from Sigma (St. Louis, MO). Tris-HCl, pH 7.4, 50 mM KCl and 2 mM EDTA at 25oC. Preparation of enzymes Removal of 3' terminal phosphate by the AS40/100 Fpg was purified chromatographically. Briefly, from the fraction crude extract of E. coli deprived of nucleic acids by To a reaction mixture containing 0.4 pmol of substrate 0.8% streptomycin, homogenous Fpg was obtained by o fractionation with ammonium sulfate, phenyl HPLC, 21-mer DNA treated with 400 units of Fpg at 25 C for 90 min, 200 g of AS 40/100 fraction was added, DEAE HPLC, gel filtration, phenyl HPLC, heparin af- µ and the mixture was incubated at 25oC for 90 min. finity HPLC and hydroxylapatite HPLC applied se- quentially. One unit of enzyme activity was defined 50 µl of distilled water and 100 µl of phenol:chloro- form (1:1) were then added, vigorously mixed and as the activity required to cleave 1 fmol of double o centrifuged at 12,000 g for 5 min, and 50 l of the stranded DNA containing 8-oxo-G at 37 C for 60 min µ (Chung et al., 1991) supernatant was then collected and dried using a vacuum concentrator (Savant, NY). AS 40/100 fraction: ammonium sulfate was added to a final concentration of 40% to the crude extract devoid of nucleic acids with stirring at 4oC for 1 h. Insertion of dG by DNA polymerase and After centrifugation at 25,000 g for 30 min, the su- sealing of the nick by DNA ligase pernatant was dialyzed against a buffer (50 mM The above 21 mer substrate DNA gapped by suc- Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.2 mM dithiothreitol cessive treatment with Fpg and the AS 40/100 frac- and 10% glycerol). The dialysate was centrifuged and tion was incubated with 1 unit of T4 DNA polymerase, the supernatant (AS 40/100 fraction) was used in the 3 units of T4 DNA ligase, 50 mg of gene 32 product, experiment. 5 mM of dNTPs and 10 mM ATP in 100 µl of a reaction buffer containing 5 mM MgCl2, 2 mM di- o Labeling of substrate double-stranded DNA thiothreitol and 100 mM Tris-HCl, at 4 C for 5 min, o o and other oligonucleotides 25 C for 5 min and 36 C for 60 min. For BER synthesis, 21-mer (5' CAGCCAATCAGT- GoxoCACCATCC 3'; Goxo=8-oxo-G) was used (a kind Visualization of enzyme products gift from Dr. Kasai at the University of Occupational To visualize products at each step, reaction mixtures and Environmental Health, Japan). The 21-mer oli- were dried in a vacuum concentrator after phenol- gonucleotide was labeled at the 5' end with [γ-32P] chloroform extraction. The dried reaction mixtures were ATP and T4 polynucleotide kinase, and then annealed dissolved in loading buffer (80% formamide, 0.1% to its complementary strand as described previously xylene cyanol and 0.1% bromophenol blue) and the (Chung et al., 1991). To confirm incision site 3' to products were separated in 8 M urea-denaturing 20% 8-oxo-G by Fpg, 46-mer oligonucleotide (5' CAGCCA- polyacrylamide gels at 2,000 V for 3 h. Gels were 108 Exp. Mol. Med. Vol. 35(2), 106-112, 2003 autoradiographed with X-Omat film (Kodak). DNA was incubated with either Fpg or the AS 40/100 fraction. The nicked product by Fpg (Figure 2, lane 2) co-migrated with the fragment produced by hot Results piperidine (lane 4), which was known to result in the 5' terminal phosphate at the gap (Chung et al., 1992).