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Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA
Introduction
Materials And Methods
Strains
Preparation of DNA substrates for in vitro repair
In vitro NER
In vitro BER
Incision assays in yeast extracts
Sensitivity to MMS
Results
BER of uracil-containing DNA is deficient in rad27 mutant extracts
Apn1 activity is reduced in rad27 mutant extracts
Apn1 endonuclease sensitizes rad27 mutant cells to MMS
BER of oxidative damage does not require Rad27
Discussion
Acknowledgements
References
Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA
ABSTRACT
INTRODUCTION
Excision repair of DNA functions to remove DNA lesions, thus suppressing mutagenesis and carcinogenesis induced by DNA damaging agents. Depending on whether the modified base is removed as a free base or an oligonucleotide, excision repair is divided into two major mechanisms: base excision repair (BER) and nucleotide excision repair (NER), respectively. BER is a major repair mechanism for endogenous DNA damage such as uracil residues in DNA, apurinic/apyrimidinic (AP) sites and oxidative damage. NER is a versatile repair pathway in that it is capable of removing a variety of DNA lesions such as those induced by UV, cisplatin, acetylaminofluorene (AAF) and polycyclic aromatic hydrocarbons (1-3).
The NER pathway can be conceptually divided into five biochemical steps: damage recognition, incision, excision, repair synthesis and DNA ligation. Over 20 NER proteins have been identified and in vitro reconstitution from purified components has been achieved for transcription-independent NER (global genome repair) (4-6). BER is initiated with direct base removal by a specific DNA glycosylase. Two classes of DNA glycosylases have been identified: glycosylases with or without associated AP lyase activities. The downstream repair events are dictated by the initiating glycosylase. After excision by a glycosylase without associated AP lyase (e.g., repair of a uracil residue in DNA), an AP site is formed in DNA. The AP site is incised at its 5[prime] side by an endonuclease, generating a 3[prime]-hydroxyl group and a 5[prime]-deoxyribose phosphate moiety. In yeast, this reaction is catalyzed by the Apn1 endonuclease (7). After removal of the 5[prime]-deoxyribose phosphate moiety and DNA repair synthesis, BER completes with a DNA ligation step (7-10). AP sites formed as a result of depurination or depyrimidination can also be repaired by this BER pathway. If BER is initiated by a glycosylase with associated AP lyase (e.g., repair of a thymine glycol residue in DNA), the resulting AP site is cleaved at its 3[prime] side by the AP lyase activity immediately after base excision (11-16). Thus, base excision and AP site cleavage reactions are concertedly mediated by a single protein. The 3[prime] baseless sugar phosphate at the DNA nick is subsequently removed, and the repair is completed by sequential DNA repair synthesis and ligation.
In yeast, NER requires the nuclease activity of Rad2 protein (17,18). Another member of the Rad2 nuclease family is Rad27 (Rth1) (19-21). Rad27 possesses a 5[prime]->3[prime] exonuclease activity and a flap endonuclease activity (21,22). Inactivation of Rad27 results in a temperature-sensitive phenotype for growth, a strong mutator phenotype, chromosome instability and a severe sensitivity to methylmethane sulfonate (MMS) (19,20,23). Apparently, Rad27 protein plays multiple roles in cells: processing Okazaki fragments during DNA replication (24-28), maintaining the stability of DNA sequences flanked by short direct repeats (29) and stabilizing micro- and minisatellite DNA sequences (30). The MMS sensitivity suggests that Rad27 may play a role in DNA repair. However, the precise role of Rad27 in yeast DNA repair is not known. Recently, it was shown that FEN1, the Rad27 homolog in higher eukaryotes, is involved in the repair of oxidized AP sites in human cell extracts (31) and a synthetic AP site analog (3-hydroxy-2-hydroxymethyltetrahydrofuran) in a Xenopus laevis in vitro BER system (32).
Rad27 is required to maintain cellular resistance to MMS, but not ionizing radiation, and only plays a minor role against UV-induced cytotoxicity (19). The mechanism responsible for this apparent specificity is unkown. By studying the role of Rad27 in different modes of DNA excision repair, we wish to gain insights into the cellular function of Rad27 in response to various DNA damaging agents. In this report, we (i) demonstrate that Rad27 is specifically required for yeast BER of AP sites in DNA; (ii) show that Apn1 endonuclease sensitizes rad27 mutant cells to MMS; and (iii) offer a molecular mechanism for the differential response of rad27 mutant cells to various DNA damaging agents.
MATERIALS AND METHODS
Strains
The Saccharomyces cerevisiae wild-type strains used were SX46A (33) and JJ567 (34). The apn1 deletion mutants were DRY370 (35) and SX46Aapn1[Delta] (MATa apn1::URA3 ade2 his3-532 trp1-289 ura3-52). The rad10 and rad27 deletion mutants were BJ2168rad10[Delta] (36) and SX46Arad27[Delta] (19), respectively.
Preparation of DNA substrates for in vitro repair
To prepare AAF-modified DNA, plasmid pUC18 (100 µg) was treated with 3 µM N-acetoxy-2-acetylaminofluorene (activated form of AAF) in the dark for 3 h in 1 ml TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) containing 20% ethanol. Damaged DNA was then purified by centrifugation in a linear 5-20% sucrose gradient as previously described (37). The resulting DNA contained at least five AAF adducts per pUC18 molecule on average (37).
Osmium tetroxide-damaged DNA was obtained by incubating the agent (300 µg/ml) with plasmid pUC18 (100 µg) in 300 µl of TES buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 100 mM NaCl) at 70°C for 90 min. Damaged DNA was then purified by centrifugation in a linear 5-20% sucrose gradient to remove nicked plasmid DNA as previously described (37). This treatment yielded approximately five damaged bases per pUC18 DNA molecule as determined by sensitive sites to Escherichia coli endonuclease III (37).
To prepare uracil-containing pUC18 DNA, the plasmid was propagated in E.coli CJ236 (ung- dut-). Plasmid DNA was then isolated and purified by alkaline lysis and CsCl-ethidium bromide equilibrium centrifugation. The resulting plasmid DNA contained randomly distributed uracil residues in place of some thymine residues.
Single-stranded oligonucleotide (30mer) containing a uracil residue at position 13 and its complementary strand were synthesized in a DNA synthesizer and purified by HPLC (Operon, Inc.). The two oligonucleotide strands were then annealed to form a duplex by incubating equal molar amounts of both oligonucleotides for 5 min at 85°C in TES buffer followed by slow cooling to room temperature. The sequence of the uracil-containing oligonucleotide is 5[prime]-GGATGGCATGCAUTAACCGGAGGCCGCGCG-3[prime].
In vitro NER
Yeast whole cell extracts for in vitro NER were prepared as previously described (38). Standard NER reaction mixture (50 µl) contained 200 ng each of AAF-pUC18 DNA and undamaged pGEM3zf DNA, 45 mM HEPES-KOH (pH 7.8), 7.4 mM MgCl2, 0.9 mM dithiothreitol, 0.4 mM EDTA, 2 mM ATP, 20 µM each dATP, dGTP and dTTP, 4 µM dCTP, 1 µCi of [[alpha]-32P]dCTP (3000 Ci/mmol), 40 mM phosphocreatine (disodium salt), 2.5 µg of creatine phosphokinase, 4% glycerol, 100 µg/ml bovine serum albumin, 5% polyethylene glycol 8000 and 300 µg of yeast cell-free extracts. After incubation at 26°C for 2 h, plasmid DNA was purified, linearized with HindIII restriction endonuclease, separated by 1% agarose gel electrophoresis and autoradiographed as described previously (37,38). To examine the DNA ligation step of NER, repaired plasmid DNA was purified and loaded directly onto a 1% agarose gel without prior linearization with HindIII. Electrophoresis was performed in the presence of 0.5% ethidium bromide.
In vitro BER
The same extracts used for in vitro NER were also used for in vitro BER and were prepared as previously described (36,37). Standard repair synthesis assays (50 µl each) contained 2 pmol of the 30mer duplex DNA, 45 mM HEPES-KOH (pH 7.8), 7.4 mM MgCl2, 0.9 mM dithiothreitol, 0.4 mM EDTA, 2 mM ATP, 20 µM each dATP, dGTP and dCTP, 4 µM dTTP, 1 µCi of [[alpha]-32P]dTTP (3000 Ci/mmol), 40 mM phosphocreatine (disodium salt), 2.5 µg of creatine phosphokinase, 4% glycerol, 100 µg/ml bovine serum albumin and 50-80 µg of yeast whole cell extracts. After incubation at 23°C for 2 h, DNA was extracted by phenol/chloroform and precipitated in ethanol. Repair products were separated by electrophoresis on a 20% denaturing polyacrylamide gel. Repair synthesis was visualized by autoradiography of the wet gel. For BER assays of plasmid DNA, the reaction buffer was identical to that of the 30mer oligonucleotide repair. After incubation at 30°C for 2 h in 80 µg of yeast extracts, DNA purification, treatments and product detection were identically performed as in NER assays described above.
Incision assays in yeast extracts
Prior to incision assays, the uracil-containing strand of the oligonucleotide duplex DNA was labeled at its 5[prime] or 3[prime] end with 32P. For 5[prime] labeling, the 30mer oligonucleotide was labeled by [[gamma]-32P]ATP with T4 polynucleotide kinase, followed by annealing to its complementary strand as described above. For 3[prime] labeling, a 31mer oligonucleotide was synthesized which was complementary to the uracil-containing strand but with an extra A at its 5[prime] end. After annealing the two oligonucleotides, a [32P]dTMP was added to the 3[prime] of the uracil-containing strand by the Klenow fragment of E.coli DNA polymerase I. The reaction mixture (20 µl) contained 50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 µM dTTP, 75 µCi of [[alpha]-32P]dTTP (3000 Ci/mmol), 10 pmol duplex DNA and 3 U Klenow fragment of E.coli DNA polymerase I. After incubation at 30°C for 30 min, DNA was extracted by phenol/chloroform and purified by a Push column (Stratagene, Inc.)
Standard incision assays were performed in a 50 µl reaction mixture containing the 32P-labeled duplex DNA (0.5 pmol), 45 mM HEPES-KOH (pH 7.8), 7.4 mM MgCl2, 0.9 mM dithiothreitol, 0.4 mM EDTA, 40 mM phosphocreatine (disodium salt), 4% glycerol, 100 µg/ml bovine serum albumin and 50-80 µg of yeast extracts. After incision reactions at 23°C for various times as indicated, a 5 µl aliquot was used for analysis by electrophoresis on a 20% denaturing polyacrylamide gel. Incision products were detected by autoradiography of the wet gel.
Sensitivity to MMS
Yeast cells grown at 30°C to stationary phase in minimummedia were harvested by centrifugation, washed with water and resuspended in 100 mM potassium phosphate (pH 7.0)(4 OD600 cells/ml). Cells were then treated with various doses of MMS as indicated at 30°C for 30 min with shaking, followed by centrifugation and washing with water. After dilution, cells were plated in minimum medium plates. Surviving colonies were counted after incubation at 30°C for 2-4 days.
Figure 1. BER of uracil-containing DNA in rad27 mutant extracts. Standard repair synthesis assays of BER were performed in rad27 or its isogenic wild-type cell extracts. (A) BER of uracil-containing plasmid pUC18 DNA. In vitro repair was at 30°C for 2 h in 80 µg of the extracts and the repair products were separated by electrophoresis on a 1% agarose gel. Reduction of the linear U-pUC18 DNA (2.7 kb) on ethidium bromide-strained gel resulted from extensive DNA fragmentation initiated by uracil-DNA glycosylase in the extracts. +Uracil, modified DNA; -Uracil, unmodified DNA. Top, ethidium bromide-stained gel; bottom, autoradiogram. (B) BER of the 30mer duplex DNA containing a uracil residue at position 13. In vitro repair was at 23°C for 2 h in 50 µg of the extracts and the repair products were separated by electrophoresis on a 20% denaturing polyacrylamide gel. Size markers in nucleotides are indicated on the right. Figure 2. AP site incisions in rad27 mutant extracts. The 30mer duplex DNA was labeled with 32P at the 5[prime] end of the uracil-containing strand. Incision assays were then performed in 80 µg cell extracts without ATP and dNTPs at 23°C for various times as indicated. Incision products were analyzed by electrophoresis on a 20% denaturing polyacrylamide gel and visualized by autoradiography. Products of 3[prime] or 5[prime] incisions at the AP site were identified (7) and are indicated by the arrowheads. Size markers in nucleotides are indicated on the left. BER of uracil residues in DNA is initiated by uracil-DNA glycosylase without associated AP lyase (7,12). In this mode of BER, AP sites are formed as repair intermediates following the glycosylase action. Subsequently, DNA incision 5[prime] to the AP site requires another enzyme, Apn1 endonuclease in yeast (7). Thus, this pathway is also a major mechanism for the repair of spontaneous or induced AP sites in DNA. To examine the role of Rad27 in this mode of excision repair, we performed in vitro repair in yeast cell-free extracts using plasmid pUC18 DNA containing uracil residues in place of some thymine residues. Under the conditions used, repair of uracil-containing DNA in yeast cell extracts is specifically mediated by the BER pathway and exclusively initiated by uracil-DNA glycosylase (7). As previously observed (39), incubation of uracil-containing pUC18 in wild-type yeast extracts led to DNA fragmentation due to extensive DNA strand breaks after sequential uracil removal and AP site cleavage (Fig. Fragmentation of uracil-containing DNA in yeast cell extracts is initiated by uracil-DNA glycosylase. Such DNA fragmentation was also observed in rad27 mutant extracts (Fig. The site-specific uracil-containing DNA was labeled at its 5[prime] end with 32P. Incision was then performed for various times in yeast extracts without ATP and dNTPs (Fig. In yeast, APN1 and RAD27 are two adjacent genes on chromosome XI. Deleting the RAD27 gene did not lead to fortuitous inactivation of the APN1 gene in this rad27 strain as indicated by Reagan et al. (19). Furthermore, we detected APN1 transcripts in rad27 mutant cells by RT-PCR (data not shown). These results indicate that the Apn1 activity is reduced in rad27 mutant extracts, thus leading to deficient repair synthesis of uracil BER (Fig. Figure 3. MMS sensitivity. Yeast cells were treated with various doses of MMS as indicated at 30°C for 30 min as described in Materials and Methods. Survival rates are expressed relative to those of the untreated cells. All strains are isogenic to the wild-type (WT) SX46A. The plasmid pGAL-APN1 contains the APN1 gene under the GAL1 promoter control. Overexpression of APN1 was induced by 1% galactose in minimum media containing 1% glucose. This overexpression did not significantly affect MMS sensitivity in wild-type cells. Results are the averages for duplicate experiments. To determine whether the MMS sensitivity of rad27 could be accounted for by deficient Apn1 activity, we quantitatively compared the sensitivity of rad27 with that of apn1 mutants. As reported by Reagan et al. (19), rad27 mutant cells were highly sensitive to MMS (Fig. Figure 4. Processing of the 5[prime] strand break in yeast extracts after DNA incision by an AP endonuclease. A 31mer duplex DNA containing a site-specific uracil residue at position 13 was labeled with 32P at the 3[prime] end of the uracil-containing strand. After incubating the DNA with E.coli uracil-DNA glycosylase (1 U) and endonuclease IV (2.5 ng) at 30°C for 30 min to cleave the AP site at the 5[prime] side, the repair intermediate containing 5[prime]-deoxyribose phosphate component was processed in yeast cell extracts (80 µg) without ATP and dNTPs at 23°C for various times as indicated. (A) Repair in wild-type extracts. (B) Repair in rad27 mutant extracts. Repair products were analyzed by electrophoresis on a 20% denaturing polyacrylamide gel. The endonuclease IV-cleaved 3[prime] DNA fragment migrated at the 18mer position (lane 1). Under this electrophoresis condition, a labeled 3[prime] DNA fragment with or without the 5[prime]-deoxyribose phosphate moiety migrated at the same position. Size markers in nucleotides are indicated on the sides. Inactivating the APN1 gene, however, did not suppress the temperature-sensitive growth of rad27 mutant cells at 37°C (data not shown), suggesting that the molecular bases for MMS sensitivity and temperature-sensitive growth phenotypes are different. These results demonstrate that Apn1 endonuclease sensitizes rad27 mutant cells to the killing effect of MMS. Incision by AP endonuclease generates a DNA strand break with a 3[prime] hydroxyl group and a 5[prime] deoxyribose phosphate moiety (40). Yeast DNA polymerase [beta] does not play a significant role in BER (41,42; X.Wu and Z.Wang, unpublished results), thus excluding the possibility of removing the 5[prime] deoxyribose phosphate by the dRPase activity of the polymerase. Based on studies of the FEN1 protein (a Rad27 homolog) in higher eukaryotes (31,32,43), we suspected that the 5[prime] deoxyribose phosphate moiety may be removed by Rad27 in yeast through its nuclease activities. To test this possibility, we examined the repair step after DNA incision. We first labeled the site-specific uracil-containing DNA at its 3[prime] end by enzymatic addition of a [32P]dTMP. The DNA was treated with E.coli uracil-DNA glycosylase and endonuclease IV, an Apn1 homolog (44), to generate a DNA strand break with a 5[prime] deoxyribose phosphate moiety. This repair intermediate was then further repaired in yeast cell-free extracts without ATP and dNTPs to avoid repair synthesis. After incubation in wild-type yeast extracts, DNA bands of 13-17 nt were evident (Fig. Figure 5. BER of osmium tetroxide-damaged plasmid DNA in rad27 mutant extracts. In vitro BER was performed in rad27 or its isogenic wild-type cell extracts for 2 h at various temperatures as indicated. (A) Standard repair synthesis assays with damaged (+OsO4) and undamaged (-OsO4) DNA. The repaired products were linearized by HindIII digestion prior to gel electrophoresis. (B) Examination of the DNA ligation step of BER. Repair reactions were similarly performed as in (A), but using only the OsO4-damaged pUC18. The repaired products were not treated with HindIII prior to electrophoresis on agarose gel containing 0.5 µg/ml ethidium bromide. OC, opened circular DNA; CC, closed circular DNA. Top, ethidium bromide-stained gel; bottom, autoradiogram. MMS treatment generates significant AP sites in DNA, which result from the removal of methylated bases by 3-methyl adenine-DNA glycosylase (Mag1) (45) and spontaneous depurination of the labile N3-methyl adenine and N7-methyl guanine (46-48). In rad27 mutant cells, unprocessed DNA strand breaks may persist at some AP sites due to incisions by the residual Apn1 activity and deficiency in subsequent processing of the 5[prime] strand breaks. Hence, we conclude that the extreme sensitivity of rad27 mutant cells to MMS is caused by unprocessed DNA strand breaks accumulated during AP site repair, which can block replication and thus lead to lethality. BER of oxidative damage is initiated by DNA glycosylases with associated AP lyase activities (7,12). This mode of BER differs from the repair of uracil residues or AP sites in DNA in that a DNA strand break is formed 3[prime] to the AP site after the combined glycosylase and AP lyase reactions. Hence, AP site does not exist as a repair intermediate. The role of Rad27 in the repair of oxidative damage was investigated. Figure 6. NER in rad27 mutant extracts. In vitro NER was performed in yeast cell-free extracts of JJ567 (lane 1), BJ2168rad10[Delta] (lane 2), SX46A (lane 3), KG119 (lane 4) and SX46Arad27[Delta] (lane 5) at 26°C for 2 h. In lanes 1-4, repair products were linearized with HindIII restriction endonuclease according to the standard repair synthesis assay. In lane 5, repair products were directly loaded onto the gel without prior treatment with HindIII to separate closed circular DNA (migrated faster) from nicked circular DNA (migrated slower). +AAF, damaged DNA; -AAF, undamaged DNA. Top, ethidium bromide-stained gel; bottom, autoradiogram. Osmium tetroxide-damaged DNA contains predominantly thymine glycols which are repaired by the Ntg proteins through the BER pathway in yeast cell-free extracts (11,14,36,37). Consistent with our previous results, BER repair synthesis (labeled by 32P) of osmium tetroxide-damaged DNA was readily detected in wild-type yeast extracts (Fig. The effect of Rad27 on NER was also determined. Under the conditions used, repair of pUC18 DNA containing AAF adducts is specifically mediated by the NER pathway in yeast cell-free extracts (37,38). In vitro NER was traced by 32P-labeling of the repair patch. In vitro NER was proficient in wild-type yeast extracts, but defective in NER-deficient rad10 or ssl2 mutant extracts (Fig. There are two major modes of BER. One BER pathway is represented by the repair of oxidative damage which is initiated by a DNA glycosylase with associated AP lyase. AP sites formed after glycosylase action are immediately cleaved at their 3[prime] side by the AP lyase activity of the same protein. The other mode of BER is represented by the repair of uracil residues and AP sites in DNA, which does not involve an AP lyase. In yeast, the protein requirements for the two BER pathways have not been completely defined, although much is known about uracil BER (7,39). Using a yeast cell-free system, we have shown that BER of uracil residues in DNA is deficient in rad27 mutant extracts, whereas BER of thymine glycols and NER of AAF are not affected by the absence of Rad27 protein. The requirements for Rad27 protein in uracil BER in vitro are 2-fold. First, in the absence of Rad27 protein, the Apn1 endonuclease activity was significantly reduced. This led to deficient AP site incision which in turn resulted in deficient repair synthesis. Secondly, following Apn1 incision, hydrolysis of one or a few nucleotides (presumably containing the 5[prime]-deoxyribose phosphate moiety) from the 5[prime] of the DNA strand break was deficient without Rad27 protein. Hence, Rad27 is specifically required for yeast BER of AP sites in DNA. The precise molecular basis for the effect of Rad27 on Apn1 activity is presently unknown. Since APN1 gene is adjacent to RAD27 gene, rad27 deletion was carefully targeted ~40 bp downstream of the RAD27 stop codon, but ~230 bp upstream of the APN1 start codon (19). Thus, the APN1 gene is expected to be intact in this rad27 mutant strain. Our results and those of Reagan et al. (19) support this conclusion. It is possible that these two functionally-related genes may be coordinately regulated. Consistent with such a possibility, we observed a 3-fold reduction in APN1 mRNA in rad27 mutant cells as measured by quantitative RT-PCR. It is also possible that the stability and/or activity of Apn1 may be directly affected by Rad27 protein. During BER of AP sites, DNA strand breaks are introduced by Apn1 incisions. Subsequent repair involves Rad27 to process the 5[prime] DNA strand breaks through nucleotide hydrolyses. Without Rad27, unprocessed DNA strand breaks may accumulate at some AP sites, which in turn can lead to lethality during DNA replication. This provides a molecular basis for the response of rad27 mutants to DNA damaging agents: highly sensitive to MMS, but not to ionizing radiation, and only slightly sensitive to UV (19). While the latter two agents cause significant DNA double strand breaks and bulky photoproducts, respectively, which are predominantly repaired by recombination and NER, respectively, MMS leads to AP sites in DNA. This mechanism of MMS sensitivity is further supported by the observation that deleting APN1 gene to prevent 5[prime] AP site incision largely restored the resistance of rad27 cells to MMS. The apn1 rad27 double mutant was slightly more sensitive to MMS than apn1 single mutant (Fig. In higher eukaryotes, it appears that the 5[prime] deoxyribose phosphate moiety derived from a natural AP site is mainly removed by the dRPase activity of DNA polymerase [beta], independent of FEN1 (8-10,31). Apparently, this polymerase [beta]-dependent mechanism is not functional in yeast (41,42; X.Wu and Z.Wang, unpublished results). Our studies suggest that the 5[prime] deoxyribose phosphate component is likely removed in yeast by hydrolysis of 1-5 nt 3[prime] to the baseless sugar phosphate. This activity of Rad27 can occur in the absence of DNA repair synthesis. Since flap DNA structure is not possible without strand displacement synthesis, the removal of nucleotides from 5[prime] of the strand break by Rad27 must be mediated by its exonuclease activity. It is possible that Rad27 may also be involved in removing DNA flap structures that are formed by repair synthesis in the presence of 5[prime]-deoxyribose phosphate moiety. Removal of the 5[prime] baseless sugar phosphate by the nuclease activities of FEN1 has been observed with modified AP sites in Xenopus and a human cell-free system (31,32). The major DNA glycosylases for oxidative damage identified so far all contain an associated AP lyase activity. Thus, repair of oxidative damage involves 3[prime] AP site incision and subsequent removal of the 3[prime] baseless sugar phosphate component. Since we did not observe a significant defect during repair of thymine glycols induced by osmium tetroxide in rad27 mutant extracts, it is likely that repair of the 3[prime] baseless moiety may involve other protein(s) in addition to the endonuclease Apn1. Supporting this notion, we detected significant residual repair synthesis with osmium tetroxide-damaged DNA in apn1 mutant extracts (7). In BER initiated by a glycosylase without associated AP lyase, removal of the 5[prime]-deoxyribose phosphate component is a rate limiting step (7). Hence, repair of oxidative damage with AP lyase should effectively avoid competing against BER of other DNA damage such as uracil residues and AP sites at the same rate-limiting step. Diverting BER into two major repair pathways with two classes of DNA glycosylases would therefore enhance the overall repair efficiencies for various lesions in the genome. We thank Grigory Dianov, Errol Friedberg and Thomas Donahue for providing us with purified E.coli endonuclease IV, yeast strains SX46Arad27[Delta] and KG119, respectively. We thank Bruce Demple for the apn1 deletion plasmid construct and yeast strain DRY370. We thank Elena Braithwaite for critical review of this manuscript. These studies were supported by a research grant CA67978 from NIH and a start-up fund from the University of Kentucky. 
RESULTS
BER of uracil-containing DNA is deficient in rad27 mutant extracts
Apn1 activity is reduced in rad27 mutant extracts
Apn1 endonuclease sensitizes rad27 mutant cells to MMS
BER of oxidative damage does not require Rad27
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
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