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J. Biol. Chem., Vol. 277, Issue 24, 21300-21305, June 14, 2002

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Repair of Clustered DNA Lesions

SEQUENCE-SPECIFIC INHIBITION OF LONG-PATCH BASE EXCISION REPAIR BY 8-OXOGUANINE^*

Helen Budworth^§, Irina I. Dianova, Vladimir N. Podust^¶, and Grigory L. Dianov^**

From the  Medical Research Council Radiation and Genome Stability Unit, Harwell, Oxfordshire OX11 0RD, United Kingdom, the ^¶ Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232 and the ^§ Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

Received for publication, February 26, 2002, and in revised form, March 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ionizing radiation induces clustered DNA damage where two or more lesions are located proximal to each other on the same or opposite DNA strands. It has been suggested that individual lesions within a cluster are removed sequentially and that the presence of a vicinal lesion(s) may affect the rate and fidelity of DNA repair. In this study, we addressed the question of how 8-oxoguanine located opposite to normal or reduced abasic sites would affect the repair of these sites by the base excision repair system. We have found that an 8-oxoguanine located opposite to an abasic site does not affect either the efficiency or fidelity of repair synthesis by DNA polymerase . In contrast, an 8-oxoguanine located one nucleotide 3'-downstream of the abasic site significantly reduces both strand displacement synthesis supported by DNA polymerase  or  and cleavage by flap endonuclease of the generated flap, thus inhibiting the long-patch base excision repair pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Random energy deposition by ionizing radiation induces a wide array of different DNA lesions (1). Ionizing radiation induces damage in DNA by direct ionization and through generation of hydroxyl radicals that attack DNA, resulting in single strand breaks (SSBs)¹ and oxidative damage to sugar and base residues (2). Two or more DNA lesions of the same or different nature may be produced proximal to each other on opposite DNA strands, generally within two helical turns of the DNA. These various types of DNA damage, known as "clustered DNA lesions", may include strand breaks that contain damaged DNA termini accompanied by multiple base lesions of varying complexity. 8-oxoguanine is one of the most abundant types of oxidative base damage and is frequently found as a component of clustered lesions (3, 4). For densely ionizing radiation (such as -particles), the yield of clustered DNA damage is high, with >50% of the SSB having a vicinal lesion (3). Recently, using enzymatic methods, it was demonstrated that ionizing radiation indeed induces clustered DNA damage containing oxidized bases and that this type of clustered damage constitutes about 50-80% of the total DNA damage (4).

Base excision repair (BER) pathways and the SSB repair pathway are the major repair systems that contribute to the processing of oxidative lesions (5-7). BER involves several steps, i.e. removal of a damaged base by a DNA glycosylase, nicking of an AP site by AP endonuclease, repair synthesis, and finally the sealing of the nick by DNA ligase (8). All of these reactions may be affected by the presence of a neighboring lesion. Several groups (reviewed in Refs. 9 and 10) have extensively studied the effects of opposing or multiple tandem lesions on DNA glycosylases and AP endonucleases. However, very little is known about the effect of these lesions on subsequent BER steps. In particular, both the fidelity and efficiency of the DNA repair synthesis step, supported by DNA polymerase  (pol ) and  (pol ), may be affected by the presence of a base lesion on the template strand.

AP sites or SSBs inhibit the processing of 8-oxoguanine when they are closely located on the opposite DNA strand (11). Thus, in the course of repair, clustered lesions containing an 8-oxoguanine opposed by either another oxidized base or an abasic site (e.g. 5-hydroxycytosine or an AP site opposite to 8-oxoguanine) will be converted into a lesion consisting of 8-oxoguanine opposite to a 5'-sugar phosphate-containing SSB. Depending on the status of the 5'-sugar phosphate (normal or oxidized/reduced), this lesion can be repaired via short- or long-patch repair. Short-patch BER involves the incorporation of one nucleotide by DNA pol  (12) accompanied by removal of the 5'-sugar phosphate moiety by the same enzyme (13) and finally the rejoining of the DNA ends by DNA ligase (14). All three reactions may be affected by the presence of 8-oxoguanine in the template strand. Long-patch repair may also involve DNA pol . DNA polymerase / adds 2-4 additional nucleotides into the repair gap, displacing the 5'-sugar phosphate as part of a flap that is further removed by a flap endonuclease (FEN1), and then DNA ligase seals the DNA ends (7). In this pathway an additional lesion present in the template DNA strand may affect the efficiency of repair even if it resides several nucleotides 3'-downstream of the lesion in the opposite strand. In this study, we have employed oligonucleotide duplexes containing 8-oxoguanine located either directly opposite or one nucleotide 3' to an SSB containing a 3'-OH end and either a normal or reduced 5'-sugar phosphate end. Using these substrates and purified human BER proteins, we characterized the effect of 8-oxoguanine on the repair of clustered lesions via short- and long-patch BER.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Synthetic oligodeoxyribonucleotides purified by high performance liquid chromatography were obtained from MWG Biotech. [-³²P]dATP, [-³²P]dCTP and [-³²P]ATP (3000 Ci/mmol) were purchased from PerkinElmer Life Sciences. Recombinant human pol  was overexpressed and purified as described previously (15). Human APE1 and uracil-DNA glycosylase with 84 amino acids deleted from the amino terminus were purified as described (16, 17). DNA ligase I was a gift from A. Tomkinson.

Substrate Labeling-- A 30-mer oligonucleotide containing a uracil residue at position 20 (5'-AATAAGCTTGATCGGCCGGUCGCGGTATAT-3') was 5'-end labeled with T4 polynucleotide kinase and [-³²P]ATP. Unincorporated labeled nucleotides were removed on a Sephadex G-25 spin column.

Substrates-- To prepare the oligonucleotide duplex, labeled oligonucleotide containing a uracil residue was annealed to its complementary strand containing guanine or 8-oxoguanine at the indicated position. The equimolar solution of both oligonucleotides in TE, 100 mM KCl, was incubated at 90 °C for 3-5 min, and the solution was allowed to cool slowly to 25 °C. Prior to the assembly of the excision reaction, the DNA substrate (500 ng, 50 pmol) was pretreated with uracil-DNA glycosylase (200 ng, 6.25 pmol) in 10 mM Hepes, pH 7.9, 1 mM EDTA, and 100 mM KCl. The reaction mixture was incubated at 37 °C for 1 h. When indicated, the resulting AP site was reduced by the addition of sodium borohydride to 0.1 M and, after incubation on ice for 10 min, the reaction buffer was exchanged to TE by filtering through a Sephadex G-25 spin column.

DNA Polymerase Assay-- The reactions (10 µl) contained 45 mM Hepes, pH 7.8, 70 mM KCl, 7.5 mM MgCl₂, 0.5 mM EDTA, 1 mM dithiothreitol, 2 mM ATP, 20 µM each of dATP, dGTP, dCTP, and dTTP, and a ³²P-labeled oligonucleotide substrate (10 ng, 1 pmol). The reactions were preincubated at 37 °C for 5 min. with 5 ng (128 fmol) of APE1, and then pol  or pol  was added at the amount indicated in the figure legends. Reactions with pol  also included 100 ng (860 fmol) of proliferating cell nuclear antigen. After an additional incubation for the indicated time at 37 °C, the reaction was stopped by the addition of 10 µl of gel-loading buffer (95% formamide, 20 mM EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol). Following incubation at 80 °C for 2 min, the reaction products were separated by electrophoresis in a 20% denaturing polyacrylamide gel containing 8 M urea in 89 mM Tris-HCl, 89 mM boric acid, and 2 mM EDTA, pH 8.0.

Reconstituted BER Reaction-- The 30-mer oligonucleotide containing a single uracil residue at position 20 was 5'-end labeled and annealed to the complementary strand containing guanine or 8-oxoguanine at the indicated position. Prior to the assembly of the excision reaction, the oligonucleotide substrate (100 ng) was pretreated with uracil-DNA glycosylase (100 ng) in 10 mM Hepes, pH 7.9, 1 mM EDTA, and 70 mM KCl. The reaction mixture was incubated at 37 °C for 1 h. Because of the instability of the AP-containing DNA, the substrates were prepared just before performing the BER reactions. BER reactions were reconstituted in a reaction mixture (10 µl) that contained 45 mM Hepes, pH 7.8, 70 mM KCl, 2 mM dithiothreitol, 7.5 mM MgCl₂, 0.5 mM EDTA, 2 mM ATP, 20 µM each of the indicated dNTPs, and ³²P-labeled oligonucleotide substrate (10 ng, 1 pmol). The reactions were initiated by the addition of purified APE1, pol , and DNA ligase I at the amount indicated in the figure legends. After incubation for the indicated time at 37 °C, the reactions were stopped by the addition of 10 µl of gel-loading buffer (95% formamide, 20 mM EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol). Following incubation at 80 °C for 2 min, the reaction products were separated by electrophoresis in a 20% denaturing polyacrylamide gel.

Flap Excision Reaction-- A 28-mer oligonucleotide containing a uracil residue at position 19 (5'-AATAAGCTTGATCGGCCGUCCGCGGTAT-3') was annealed to the complementary 30-mer containing two extra nucleotides (TA) at the 5'-end and labeled by filling the protruding ends using an Escherichia coli DNA polymerase Klenow fragment in the presence of [-³²P]dATP and cold TTP. Unincorporated labeled nucleotides were removed on a Sephadex G-25 spin column. Prior to the assembly of the excision reaction, the oligonucleotide substrate (100 ng) was pretreated with uracil-DNA glycosylase (100 ng) in 10 mM Hepes, pH 7.9, 1 mM EDTA, and 70 mM KCl. The reaction mixture was incubated at 37 °C for 1h. The resulting AP site was reduced by the addition of sodium borohydride to 0.1 M and, after incubation on ice for 10 min, the reaction buffer was exchanged to TE by filtration through a Sephadex G-25 spin column. The excision reactions were reconstituted in a reaction mixture (10 µl) that contained 45 mM Hepes, pH 7.8, 70 mM KCl, 7.5 mM MgCl₂, 0.5 mM EDTA, 1 mM dithiothreitol, 2 mM ATP, 2 mg/ml bovine serum albumin, 20 µM each of dATP, dGTP, dCTP and dTTP, and a ³²P-labeled oligonucleotide substrate (10 ng, 1 pmol) and APE1, FEN1, and pol  at the amount indicated in the figure legends. The reactions were initiated by adding substrate oligonucleotides and, after incubation for the indicated time at 37 °C, the reactions were stopped by addition of 10 µl of gel-loading buffer (95% formamide, 20 mM EDTA, 0.02% bromphenol blue, and 0.02% xylene cyanol). Following incubation at 80 °C for 2 min, the reaction products were separated by electrophoresis in a 20% denaturing polyacrylamide gel. All experiments were repeated at least 3-5 times, and representative phosphorimages of the gels are shown.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

8-Oxoguanine in a Template Strand Does Not Affect Either the Efficiency or Fidelity of Short-patch DNA Repair-- During short-patch BER, after removal of a damaged base and incision of the AP site by AP endonuclease, DNA polymerase  incorporates a single nucleotide into the repair gap and removes a 5'-sugar phosphate. To accomplish repair, DNA ligase seals the DNA ends (8). To investigate whether 8-oxoguanine located within the repair gap would affect short-patch BER, we constructed a substrate oligonucleotide duplex containing a strand break with a 5'-sugar phosphate moiety opposing an 8-oxoguanine on the template strand (Fig. 1A). We first examined the DNA repair synthesis step and found that at the concentrations used in our experiments, pol  efficiently bypassed 8-oxoguanine and was also able to extend repair synthesis beyond one nucleotide (Fig. 1B). We also found that, with this substrate, dCMP was exclusively incorporated opposite to 8-oxoguanine because repair synthesis was completely blocked in the absence of dCTP, although dATP, dGTP, and TTP were added to the reaction at the normal concentration (data not shown). Moreover, in an assay where equal amounts of dCTP and dATP were used, only the incorporation of dCMP was observed (Fig. 1C). We thus conclude that 8-oxoguanine located in the repair gap does not affect either the efficiency or the fidelity of repair synthesis.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effect of 8-oxoguanine on short-patch BER. A, schematic representation of the 32P-5'-labeled (*) oligonucleotide substrate used. B, 1 pmol of the 5'-end-labeled substrate oligonucleotide duplex containing 8-oxoguanine (left panel) or the guanine (right panel) opposite to the SSB with the 5'-sugar phosphate (pR) was incubated for 20 min at 37 °C with the indicated amount of pol  in conditions described under "Experimental Procedures." After incubation, reactions were stopped by the addition of gel-loading buffer and, following incubation at 80 °C for 2 min, the reaction products were separated by electrophoresis in a 20% denaturing polyacrylamide gel. C, 1 pmol of the unlabeled substrate oligonucleotide duplex containing 8-oxoguanine opposite to the SSB with 5'-sugar phosphate (pR) was incubated for 20 min at 37 °C with the indicated amount of pol  in conditions described under "Experimental Procedures" in the presence of equal amounts (20 µM each) of dCTP and dATP, of which either dCTP (left panel) or dATP (right panel) was labeled. After incubation, reactions were stopped by the addition of a formamide-dye solution and processed as described above.

To investigate whether a neighboring 8-oxoguanine residue affects removal of the 5'-sugar phosphate or the ligation step, we reconstituted short-patch BER with purified human APE1, pol , and DNA ligase. Incubation of the substrate with APE1 resulted in a 19-mer product generated by the incision of the oligonucleotide at the AP site (Fig. 2, lane 2). DNA polymerase pol  and DNA ligase, when added to the reaction, stimulated the complete repair and restoration of the full-length 30-mer product within 20 min for both control (Fig. 2, lanes 7-10) and 8-oxoguanine-containing substrates (Fig. 2, lanes 3-6). We thus conclude that repair of the 5'-nicked AP site opposite to 8-oxoguanine was as effective as repair of the control oligonucleotide duplex containing a 5'-nicked AP site opposite to guanine.

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Short-patch BER reconstituted with purified proteins. A, schematic representation of the 32P-5'-labeled (*) oligonucleotide substrates used. B, 1 pmol of the 5'-end-labeled substrate oligonucleotide duplex containing the 8-oxoguanine (left panel) or the guanine (right panel) opposite to the abasic site (R) was incubated for the indicated time in the buffer containing magnesium, dNTPs, 60 fmol of APE1, 70 fmol of pol , and 80 fmol of DNA ligase I. Reactions were stopped by the addition of an equal volume of formamide-dye solution, and products were analyzed by electrophoresis in a 20% denaturing polyacrylamide gel. Lane 1 contains untreated substrate (UT), and lane 2 contains substrate incised (I) with APE1.

Effect of 8-Oxoguanine in a Template Strand on Long-patch DNA Repair Synthesis by pol -- To address the effect of 8-oxoguanine on long-patch repair, we used substrates containing a reduced AP site and 8-oxoguanine located opposite to it or one nucleotide downstream to the AP site (Fig. 3, A and B). Polymerase  is not able to remove a reduced 5'-sugar phosphate, and the incorporation of at least 2-4 nucleotides is required to accomplish the repair of reduced AP sites (13, 18). We found no effect of reduction of the 5'-sugar phosphate on polymerase synthesis through 8-oxoguanine located opposite to the nick (Fig. 3C); however, the position of 8-oxoguanine in the template strand had a dramatic effect on the efficiency of bypass. When 8-oxoguanine was moved one nucleotide downstream of the nick, pol  was able to insert a nucleotide opposite the base preceding 8-oxoguanine but was unable to efficiently bypass the 8-oxoguanine itself (Fig. 3D, left panel). In a control experiment with a substrate containing guanine instead of 8-oxoguanine, pol  readily generated strand displacement even at the lowest concentration used (Fig. 3D, right panel).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effect of 8-oxoguanine on long-patch repair synthesis by pol . A and B, schematic representation of the 32P-5'-labeled (*) oligonucleotide substrate used. 1 pmol of the substrate oligonucleotide duplex containing 8-oxoguanine opposite to the SSB with a reduced 5'-sugar phosphate was incubated for 20 min at 37 °C with the indicated amount of pol  in conditions described under "Experimental Procedures." After incubation, reactions were stopped by the addition of gel-loading buffer and, following incubation at 80 °C for 2 min, the reaction products were separated by electrophoresis in a 20% denaturing polyacrylamide gel. C, 1 pmol of the substrate oligonucleotide duplex containing 8-oxoguanine (left panel) or guanine (right panel) positioned one nucleotide downstream opposite to the SSB with a reduced 5'-sugar phosphate was incubated for 20 min at 37 °C with indicated amount of pol  in conditions described under "Experimental Procedures." After incubation, reactions were stopped by the addition of formamide-dye solution and processed as described above.

The presence of 8-oxoguanine on the template strand may directly affect the polymerization step itself or indirectly affect the DNA synthesis reaction by reducing the efficiency of strand displacement by DNA polymerase. To evaluate the contribution of these components to the observed repair synthesis block, we tested whether 8-oxoguanine in the same sequence would block polymerization by pol  in a primer extension reaction. We found that pol  readily bypassed 8-oxoguanine (second nucleotide in a template strand), and only a moderate reduction in the rate of addition of the nucleotide opposing 8-oxoguanine was observed (Fig. 4). These data indicate that the major delay may be attributed to the inhibition of the strand displacement reaction. If strand displacement efficiency plays a major role in the inhibitory effect of 8-oxoguanine, then the inhibitory effect should be less pronounced in an AT-rich sequence. To test this surmise, a substrate containing 8-oxoguanine in an A:T-rich sequence context was constructed (Fig. 5A). Using this substrate, we find that although 8-oxoguanine still inhibits the strand displacement reaction (Fig. 5B), the effect is not as dramatic as that with the G:C-rich substrate (compare Figs. 3D and 5B).

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Bypass of 8-oxoguanine by pol  during primer extension reaction. A, schematic representation of the 32P-5'-labeled (*) oligonucleotide substrate used. B, 1 pmol of the substrate oligonucleotide containing 8-oxoguanine was incubated for 20 min at 37 °C with the indicated amount of pol  in conditions described under "Experimental Procedures." After incubation, reactions were stopped by the addition of gel-loading buffer and, following incubation at 80 °C for 2 min, the reaction products were separated by electrophoresis in a 20% denaturing polyacrylamide gel.

View larger version (53K): [in this window] [in a new window]   Fig. 5.   Effect of sequence context on 8-oxoguanine bypass during long-patch repair synthesis by pol . 1 pmol of the 5'-end-labeled substrate oligonucleotide duplex containing 8-oxoguanine (left panel) or guanine (right panel) positioned one nucleotide downstream opposite to the SSB with a reduced 5'-sugar phosphate (A) was incubated for 20 min at 37 °C with the indicated amount of pol  in conditions described under "Experimental Procedures." After incubation, the reactions were stopped by the addition of gel-loading buffer and, following incubation at 80 °C for 2 min, the reaction products were separated by electrophoresis in a 20% denaturing polyacrylamide gel (B).

8-Oxoguanine in a Template Strand Inhibits Flap Removal by FEN1-- During long-patch BER, the incorporation of several nucleotides into the repair gap leads to the generation of a flap containing 5'-deoxyribose phosphate. The FEN1 protein alone is not able to remove 5'-deoxyribose phosphate but rather releases it as part of a small oligonucleotide (2-4-mer) (18). Impaired strand displacement caused by the presence of 8-oxoguanine in a template strand may also affect flap excision and, as a consequence, the entire long-patch BER pathway. To address this, we have 3'-end labeled a substrate containing a reduced AP site and an 8-oxoguanine one nucleotide 3'-downstream in the opposite strand (Fig. 6A) and measured the excision of the pol -generated flap by the FEN1 protein in a reconstituted system containing human APE1, pol , and FEN1 proteins. We found that, after APE cleaved the AP site (Fig. 6, Time: 5 min), the flap generated by pol  was readily removed from the control substrate containing guanine (Fig. 6, left panel). However, 8-oxoguanine in the template strand significantly reduced the rate of flap removal (Fig. 6, right panel).

View larger version (70K): [in this window] [in a new window]   Fig. 6.   Effect of 8-oxoguanine in a template strand on flap removal by FEN1. A, schematic representation of the 32P-3'-labeled (*) oligonucleotide substrate used. B, 1 pmol of the 3'-end-labeled substrate oligonucleotide duplex containing 8-oxoguanine residing one nucleotide downstream to the abasic site (R) was incubated for the indicated time in the buffer containing magnesium, dNTPs, 100 fmol of APE1, 25 fmol of pol , and 60 fmol of FEN1. Reactions were stopped by the addition of an equal volume of formamide-dye solution, and products were analyzed by electrophoresis in a 20% denaturing polyacrylamide gel.

Effect of 8-Oxoguanine in a Template Strand on Long-patch DNA Repair Synthesis by pol -- Although it was recently demonstrated that pol  is responsible for the initiation of both the short- and long-patch pathways of BER (19), either pol  or pol  may contribute to further incorporation during long-patch BER (20, 21). Thus, it was interesting to test whether 8-oxoguanine would also interfere with strand displacement by pol . Using the same oligonucleotide substrate, we found that 8-oxoguanine on the template strand also constitutes a strong block for pol . The polymerization reaction was nearly completely blocked after addition of the first nucleotide (Fig. 7, left panel), although with a control substrate we observed efficient strand displacement synthesis, resulting in complete strand displacement and accumulation of the 30-mer full-length product (Fig. 7, right panel).

View larger version (89K): [in this window] [in a new window]   Fig. 7.   Effect of 8-oxoguanine on long-patch repair synthesis by pol . A, schematic representation of the 32P-5'-labeled (*) oligonucleotide substrate used. 1 pmol of the substrate oligonucleotide duplex containing an 8-oxoguanine (left panel) or a guanine (right panel) positioned one nucleotide downstream opposite to the SSB with a reduced 5'-sugar phosphate was incubated for 20 min at 37 °C with the indicated amount of pol  in the presence of 100 ng of proliferating cell nuclear antigen. After incubation, reactions were stopped by the addition of gel-loading buffer, and following incubation at 80 °C for 2 min the reaction products were separated by electrophoresis in a 20% denaturing polyacrylamide gel.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

About 70-80% of radiation-induced DNA lesions are the result of reactive oxygen species generated by the hydrolysis of water in the vicinity of DNA (1). Although it was earlier proposed that a high density of hydroxyl radicals induced by irradiation may induce multiple closely spaced DNA lesions (23), until recently these lesions were considered as regular oxidative lesions that can be repaired by BER. The first indication of the unique nature of radiation-induced DNA damage emerged from the theoretical studies of radiation-induced track structures (24, 25). Later on, the existence of complex DNA lesions induced by radiation was demonstrated experimentally (3, 4, 26). Theoretically, these complex lesions (known as clustered lesions) may include different combinations of base lesions or SSB and base lesions. Early studies indicated that the complexity of clustered DNA lesions poses a significant challenge to repair systems and may cause infidelity of repair, resulting in mutations and inefficient repair that could potentially lead to the formation of deletions and chromosome rearrangements (27-30). In this report, we have studied the repairability of clustered lesions by different BER pathways and demonstrated that the damaging effect of such lesions depends on the sequence in which they occur as well as on the relative positioning of primary lesions within a cluster. Short-patch BER is normally accomplished by the removal and replacement of a single nucleotide (12). For this repair pathway, the major threat may be the repair of simultaneous modifications of complementary bases. The processing of one of the lesions would generate an AP site or an SSB that would block removal of the opposing modified base (31-33) and would lead to the presence of a modified base in the repair gap on the template strand. To model this scenario, we studied the repair of an AP site located opposite to 8-oxoguanine. We have found that 8-oxoguanine in a repair gap does not affect either the fidelity (dCMP is normally incorporated opposite to 8-oxoguanine) or the efficiency of repair (in a reconstituted system, the rate of repair of the AP site located opposite to guanine or 8-oxoguanine is similar). Although this report shows the first data on the tolerance of both the 5'-deoxyribose phosphate lyase activity by pol  and the efficiency of DNA ligase to the presence of 8-oxoguanine in the repair gap, the fidelity and the rate of bypass of 8-oxoguanine by pol  have been studied before by several groups (34-36). It is generally accepted now that 8-oxoguanine bypass depends on the DNA polymerase and the structure of the substrate used (34). Indeed, pol  has evolved as a distributive polymerase designed to fill one-nucleotide gaps in short-patch BER and has increased activity and fidelity on single-nucleotide gap substrates (35, 36), which is in good agreement with our data. However, when the fidelity of the bypass was measured on substrates with longer gaps or in primer extension reactions catalyzed by pol , both the efficiency and fidelity of DNA synthesis were reduced (34).

To accomplish the repair of chemically modified AP sites via long-patch BER, either pol  or pol  should incorporate at least 2 nucleotides into the repair gap before FEN 1 would be able to remove the generated two-nucleotide flap (18). As we demonstrate in this report, 8-oxoguanine in the template strand strongly inhibits strand-displacement DNA synthesis by both DNA polymerases  and  (Figs. 3D and 7B) and, as a consequence. inhibits flap excision, thus blocking long-patch BER (Fig. 6). This is an unexpected result, because a previous study (22) has shown that both pol  and pol  can bypass 8-oxoguanine reasonably well in a primer extension reaction. Indeed, we also observed a reasonable bypass of 8-oxoguanine in a primer extension reaction (Fig. 4). However, 8-oxoguanine bypass was significantly reduced under strand displacement conditions. In support of the "strand displacement model" for the explanation of the bypass block by 8-oxoguanine present in the repair gap, we demonstrated that the severity of the polymerase block depends on the sequence around 8-oxoguanine; the more G:C base pairs, the more difficult it is for DNA polymerase to unwind DNA and, at the same time, bypass 8-oxoguanine. In conclusion, as we demonstrate here, the repairability of clustered lesions significantly depends on the relative location of the primary DNA lesions as well as on the sequence context of the damaged DNA. Certain lesion combinations within a cluster that will block lesion processing by DNA repair enzymes may result in genetic instability and reduced cell viability.

	ACKNOWLEDGEMENTS

We thank David Sherratt and Peter O'Neill for fruitful discussions; Sarah Allinson and Kate Sleeth are thanked for critical reading of the manuscript and Alan Tomkinson for providing DNA ligase I.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by National Institutes of Health Grant GM52948 (to Ellen Fanning).

^** To whom correspondence should be addressed. Tel.: 44-1235-824-563; Fax: 44-1235-834-776; E-mail: g.dianov@har.mrc.ac.uk.

Published, JBC Papers in Press, March 28, 2002, DOI 10.1074/jbc.M201918200

	ABBREVIATIONS

The abbreviations used are: SSB, single-strand break; 8-oxoguanine, 8-oxo-7,8-dihydroguanine; BER, base excision repair; AP, apurinic/apyrimidinic or abasic; pol , DNA polymerase ; pol , DNA polymerase ; FEN1, flap endonuclease; APE1, apurinic/apyrimidinic endonuclease 1; TE, 10 mM Tris, pH 8.0, 1 µM EDTA.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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