Nucleic Acids Research, 2001, Vol. 29, No. 14 3123-3130
© 2001 Oxford University Press
Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts
306 Health Sciences Research Building, Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA
Received December 8, 2000; Revised and Accepted May 23, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) ß,
or 
. Base excision repair was also proficient without Polß. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Pol
(pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Pol
(pol3-1) or Pol
(pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Pol and Pol, respectively. Deleting the polymerase catalytic domain of Pol (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Pol, but not by purified yeast Pol. These results suggest that efficient repair synthesis of yeast NER requires both Pol and Pol in vitro, and that the low fidelity Pol is not accessible to repair synthesis during NER. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nucleotide excision repair (NER) is an important mechanism for removing a wide spectrum of different base lesions in DNA. In humans, defects in NER can lead to the hereditary disease xeroderma pigmentosum (XP) (1). XP patients exhibit photosensitivity and a highly increased incidence of skin cancers (1). Therefore, NER plays a crucial defensive role against cytotoxicity, mutagenesis and carcinogenesis induced by a variety of DNA damaging agents.
Conceptually, NER can be divided into five biochemical steps: damage recognition, incision, excision, repair synthesis and DNA ligation. Depending on whether the repaired DNA strand is transcribed by RNA polymerase II or not, NER is further differentiated by two subpathways: global genome repair and transcription-coupled repair (2,3). In the yeast Saccharomyces cerevisiae, the first three steps of global genome repair require at least the following proteins and protein complexes: Rad7, Rad16, Rad14, replication protein A (RPA), Rad4, Rad23, TFIIH, Rad2, Rad1 and Rad10 (4–7). In the transcription-coupled repair in yeast, Rad7 and Rad16 are replaced by the presumptive coupling factor Rad26 (8,9). The last step of yeast NER requires DNA ligase I encoded by the CDC9 gene (10). The presence of DNA ligase IV cannot substitute for the NER function of ligase I (10).
Based on an in vitro system reconstituted from purified mammalian NER proteins, the repair synthesis step of NER involves RPA, replication factor C and proliferating cell nuclear antigen (PCNA) (11,12). Either purified DNA polymerase (Pol) or Pol can fill the DNA gap in this reconstituted system (11,12). Using permeabilized human fibroblasts exposed to UV radiation, Nishida et al. (13) found that Pol is required for repair synthesis. In a separate study, Zeng et al. (14) reported that repair of UV-irradiated DNA in HeLa nuclear extract was inhibited by antibodies against human Pol. In contrast, repair synthesis of NER in oocyte extracts of Xenopus laevis was reported to require both Pol and Polß (15).
In yeast, the requirement for RPA and PCNA in NER has been demonstrated in a cell-free system (16). Yeast cells contain DNA Pol, ß, 
, , , and . Most recently, the eighth S.cerevisiae DNA polymerase was identified as the TRF4 gene product and was named Pol
(17), which is required for sister chromatid cohesion. It should be noted that this polymerase bears no relation to the recently identified human Pol encoded by the DINB1 gene (18,19). By analyzing molecular weight changes in cellular DNA after UV radiation, Budd and Campbell (20) concluded that Pol and Pol are involved in repair of UV-induced damage in yeast. However, this study did not differentiate between NER and base excision repair (BER) (20). BER has been detected with UV-irradiated DNA in yeast (21), and a requirement for Pol in yeast BER has been reported (22). Therefore, further biochemical analysis would shed more light on the DNA polymerase requirement for yeast NER.
Most recently, it was found that DNA Pol is an extraordinarily low fidelity polymerase (23–25). Furthermore, the expression of yeast Pol is induced by UV radiation (26,27). These observations raised the question of whether Pol is accessible to repair synthesis during NER. Participation or interference of Pol in repair synthesis of NER would significantly affect the repair fidelity. Using a cell-free system, we have performed biochemical analyses of yeast NER with respect to the DNA polymerase requirement for and Pol accessibility to the repair synthesis step. In this report, we show that efficient repair synthesis in yeast cell-free extracts requires both Pol and Pol, and present evidence suggesting that the low fidelity DNA Pol is not accessible to repair synthesis during yeast NER in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials
Osmium tetroxide and cis-diamminedichloroplatinum (II) (cisplatin) were purchased from Sigma (St Louis, MO). N-acetoxy-N-2-acetylaminofluorene (AAAF) was obtained from the Midwest Research Institute (Kansas City, MO). The S.cerevisiae wild-type strains used were CL1265-7C (28), SX46A (29), TC102 (30), BY4741 (MATa his3 leu2 met15 ura3) and CWY231 (MATa ade1 his2 leu2-3,112 trp1-1 bar1
ura3ns) (31). The S.cerevisiae mutant strains used were AMY32 (rev3) (28), BY4741rad30 (rad30) SK-2-1ß (pol4) (32), TAY237 (pol2-16) (31), and the temperature-sensitive mutants 488 (pol1-17) (22), YHA302 (pol2-18) (33) and ts370 (cdc2-1/pol3-1) (22).
Purified yeast DNA Pol containing associated primase activity was provided by David C. Hinkle (University of Rochester, Rochester, NY). Purified yeast Pol and Pol were obtained from Akio Sugino (Osaka University, Osaka, Japan). One unit of DNA Pol incorporates 1 nmol of total nucleotide per hour at 30°C, using activated salmon sperm DNA as the substrate. One unit of DNA Pol or Pol incorporates 1 nmol of total nucleotide per 30 min at 30°C, using poly(dA).oligo(dT) as the substrate (34). Yeast DNA Pol (Rad30 protein) was purified to near homogeneity as previously described (35).
Damaged DNA substrates
Single-stranded oligonucleotide U-mse1 (30mer) containing a uracil residue at position 13 and its complementary strand (30mer) were synthesized by Operon (Alameda, CA). The nucleotide sequence of the uracil-containing strand is 5'-GGATGGCATGCAUTAACCGGAGGCCGCGCG-3'. Equal molar amounts of the two oligonucleotides were mixed and annealed by incubating for 5 min at 85°C in TES (10 mM Tris–HCl pH 7.5, 1 mM EDTA and 100 mM NaCl) buffer followed by cooling slowly to room temperature.
To prepare cisplatin-damaged DNA, plasmid pUC18 (100 µg/ml) in TE (10 mM Tris–HCl pH 7.5 and 1 mM EDTA) buffer was incubated with cis-dichlorodiamineplatinum (II) at a drug/nucleotide ratio of 0.005 at 37°C for 20 h in the dark. After adding NaCl to 0.5 M, the DNA was purified by centrifugation in a linear 5–20% sucrose gradient as described by Wang et al. (21). Purified DNA was precipitated in ethanol, dissolved in TE buffer, and stored at –20°C. To prepare osmium tetroxide-damaged DNA, plasmid pUC18 (100 µg) was treated with the agent at 70°C for 90 min in TES buffer (300 µl). The DNA was then purified by centrifugation in a linear 5–20% sucrose gradient to remove nicked plasmids (21). To prepare DNA containing N-acetyl-2-aminofluorene (AAF) adducts, pUC18 (100 µg) was incubated at 37°C for 3 h in 1 ml of TE buffer containing 3 µM AAAF (the activated form of AAF) and 20% ethanol. The DNA was then purified by centrifugation in a linear 5–20% sucrose gradient (21).
In vitro DNA repair
Yeast cell-free extracts were prepared according to our previously reported methods (36,37). The same extracts were used for both in vitro NER and BER. However, NER and BER were carried out under different reaction conditions with different DNA lesions. In vitro NER and BER were performed as described by Wang et al. (21,36,37) and are described briefly below.
A standard NER reaction mixture (50 µl) contained 200 ng each of damaged 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 [-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 250–300 µg of yeast cell-free extracts. After incubation at 26°C for 2 h, EDTA and RNase A were added to 20 mM and 20 µg/ml, respectively, and incubated at 37°C for 10 min. SDS and proteinase K were added to 0.5% and 200 µg/ml, respectively, and incubated at 37°C for 30 min. Plasmid DNA was purified by phenol/chloroform extraction, and linearized with HindIII restriction endonuclease. DNA was separated by electrophoresis on a 1% agarose gel and repair synthesis was visualized by autoradiography.
A standard BER reaction mixture (50 µl) contained 200 ng each of osmium tetroxide-damaged pUC18 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 dCTP, 4 µM dTTP, 1 µCi of [-32P]dTTP (3000 Ci/mmol), 40 mM phosphocreatine, 2.5 µg of creatine phosphokinase, 4% glycerol, 100 µg/ml bovine serum albumin and 50 µg of yeast cell-free extracts. After incubation at 30°C for 2 h, the DNA was purified by phenol/chloroform extraction, separated by electrophoresis, and visualized by autoradiography of the gel as described above for NER assays. For BER of the uracil-containing substrate U-mse1, 2 pmol of the 30mer duplex DNA was used in place of the damaged pUC18 DNA in the standard BER assay described above, and incubated at 23°C for 2 h. Reactions were stopped by phenol/chloroform extraction and the DNA was recovered by precipitation in ethanol. Repair products were separated by electrophoresis on a 20% denaturing polyacrylamide gel. Repair synthesis was visualized by autoradiography of the wet gel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NER in yeast cell-free extracts lacking Pol
or PolYeast Pol
and Pol are two lesion bypass DNA polymerases encoded by the non-essential genes REV3 and RAD30, respectively (26–28,35,38,39). To examine whether these two DNA polymerases affect repair synthesis of yeast NER, we performed in vitro NER in rev3 and rad30 deletion mutant extracts, using plasmid DNA containing cisplatin or AAF adducts. We have shown previously that under the conditions used cisplatin and AAF DNA adducts are repaired specifically by the NER pathway in yeast cell-free extracts (6,36,37,40,41). NER was monitored by radiolabeling the repair patch during DNA repair synthesis (36,37). As shown in Figure 1A, repair synthesis of NER in cisplatin-damaged DNA was not affected by deleting the REV3 gene. Repair synthesis of NER in AAF-adducted DNA was also not affected by deleting the RAD30 gene (Fig. 1B). These results indicate that yeast Pol and Pol are not required for NER in vitro.
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Yeast Polß is not required for NER or BER in vitro
DNA Polß is an important repair polymerase for BER in mammalian cells (42). Yeast Polß encoded by the POL4 gene is not essential for growth (32,43,44). Thus, pol4 deletion mutants have been isolated (32,43,44). To examine whether Polß plays an important role in yeast NER or BER, we performed repair in pol4 deletion mutant extracts. In vitro NER assays were carried out using AAF-damaged plasmid DNA as the repair substrate. As shown in Figure 2, deleting the POL4 gene did not affect repair synthesis of NER in yeast extracts. These results suggest that Polß is not required for NER in yeast.
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Depending on whether the initiating DNA glycosylase is with or without an associated AP lyase, two modes of BER are known. For example, repair of oxidative base damage in DNA is initiated by a glycosylase with associated AP lyase, while repair of uracil residues in DNA is initiated by a glycosylase without associated AP lyase. To examine both modes of BER in yeast pol4 deletion mutant extracts we used osmium tetroxide-damaged plasmid DNA that contained thymine glycol as the major damage and uracil-containing short duplex DNA as the repair substrates. Under the conditions used, BER was specifically measured without interference by NER (45). As shown in Figure 3A, BER of osmium tetroxide-damaged DNA was not affected by deleting the POL4 gene. BER of uracil residues in DNA in yeast pol4 mutant extracts was then compared with that in two different wild-type yeast extracts. As shown in Figure 3B, uracil repair was not significantly affected without yeast Polß. These results suggest that yeast Polß is not required for BER in vitro.
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Deficient NER in pol2-18 and pol3-1 mutant extracts
To identify the DNA polymerase(s) required for yeast NER, we examined the repair synthesis step in pol1-17, pol2-18 and pol3-1 mutant extracts. The pol1-17, pol2-18 and pol3-1 mutant cells are temperature-sensitive for growth. Previously, Boulet et al. (46) showed that the Pol
activity in pol1-17 cells and the Pol activity in pol3-1 cells were reduced to undetectable levels after shifting the growth temperature from 24 to 36°C for 2 h. Araki et al. (33) showed that partially purified mutant Pol (pol2-18) was very sensitive to temperature shift with a half-life of <1 min at 45°C. Therefore, the pol1-17, pol2-18 and pol3-1 mutant cells were grown at 23°C to late logarithmic phase and then at 37°C for 2 h before preparation of cell-free extracts. We expected that this growth condition should lead to inactivation of the mutant DNA polymerases in the corresponding mutant cells. To validate this expectation, we determined the effect of the temperature shift on the growth of these mutant cells. One hour after shifting to 37°C, all three mutants stopped growing. From 1 to 2 h at 37°C, viable cells of pol1-17, pol2-18 and pol3-1 were slightly reduced from 8.4 x 107 to 8.2 x 107, 2.2 x 107 to 1.7 x 107, and 2.3 x 107 to 1.9 x 107 per ml, respectively. In contrast, from 1 to 2 h at 37°C, wild-type cells continued to grow from 3.2 x 107 to 4.2 x 107 per ml. These results suggest that the temperature-sensitive Pol, and were inactivated in the mutant cells after incubation for 2 h at 37°C.
NER of AAF adducts in plasmid DNA was performed in the mutant cell extracts at 23°C. As shown in Figure 4 (lanes 1 and 2), repair synthesis was readily detected in wild-type or pol1-17 mutant extracts. In contrast, repair synthesis activities in pol2-18 (Fig. 4, lane 3) and pol3-1 (Fig. 4, lane 4) mutant extracts were significantly reduced. However, residual repair synthesis was observed in both pol2-18 and pol3-1 mutant extracts when compared with the undamaged internal control (Figs 4, lanes 3 and 4, 5A, lanes 2 and 5, and 5B, lanes 2 and 7). To show direct participation of DNA Pol and in yeast NER, we complemented pol2-18 and pol3-1 mutant extracts with the purified yeast polymerases. Deficient repair synthesis in pol2-18 mutant extracts was partially complemented by 0.05 U of purified yeast Pol (Fig. 5A, lane 3). In contrast, purified yeast Pol (80 U) had no effect (Fig. 5A, lane 4). Similarly, deficient repair synthesis in pol3-1 mutant extracts was partially complemented by 0.25 U of purified yeast Pol (Fig. 5A, lane 6), but not by purified yeast Pol (80 U) (Fig. 5A, lane 7). Furthermore, the catalytic subunit of Pol alone was also able to partially complement pol2-18 mutant extracts (Fig. 5B, lanes 3 and 4), but not pol3-1 mutant extracts (Fig. 5B, lanes 5 and 6). The catalytic subunit of Pol alone was able to partially complement pol3-1 mutant extracts (Fig. 5B, lanes 10 and 11), but not pol2-18 mutant extracts (Fig. 5B, lanes 8 and 9). These results suggest that both Pol and Pol are required for yeast NER in vitro.
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Participation of both Pol
and Pol in yeast NER raised the question of whether complete filling of each DNA gap requires both polymerases. To address this question, we performed NER reaction in pol2-18 and pol3-1 mutant extracts. After repair, the plasmid DNA was purified and loaded directly onto a 1% agarose gel without prior digestion with the HindIII restriction endonuclease. This modification of the standard in vitro NER assay allowed examination of the ligation step of the repair (10). If the DNA gap was not completely filled, subsequent DNA ligation would not occur. As expected for NER in the wild-type extracts, the vast majority of the repair patches were in the ligated form of the plasmid DNA (Fig. 6, lane 1). Similarly, the repair patches synthesized by the residual repair activities in pol2-18 and pol3-1 mutant extracts were mostly contained in the ligated form of the plasmid (Fig. 6, lanes 2 and 3). These results indicate that some DNA gaps were completely filled during residual NER in the mutant extracts. Therefore, we conclude that complete synthesis of a DNA gap during yeast NER can be performed by either Pol or Pol, although repair synthesis is more efficient when both polymerases are present.
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The catalytic domain of yeast Pol
is required for NER in vitroRecently, Kesti et al. (31) showed that the catalytic domain of Pol
is dispensable for viability of yeast cells. To determine whether the catalytic domain of Pol is also dispensable for NER, we performed repair assays in pol2-16 mutant extracts. The sequence encoding amino acids 176–1134 of yeast Pol that contains the polymerase and the 3'
5' exonuclease functions has been deleted in pol2-16 mutant cells (31). As shown in Figure 7 (compare lanes 1 and 2), repair synthesis in pol2-16 mutant extracts was deficient. Again, residual repair synthesis activity in the mutant extracts was observed (Fig. 7, lane 2). Furthermore, the repair patches synthesized by the residual repair activity in pol2-16 mutant extracts were mostly contained in the ligated form of the plasmid DNA (Fig. 7, lane 2). The deficient repair synthesis in pol2-16 mutant extracts was complemented by 0.08 U of purified yeast Pol (Fig. 7, lane 4). In contrast, 0.4 U of purified yeast Pol was unable to complement the deficient repair synthesis in pol2-16 mutant extracts (Fig. 7, lane 3). These results show that the catalytic domain of yeast Pol is required for NER in vitro.
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The low fidelity Pol
is not accessible to repair synthesis of NER in yeast extractsPol
synthesizes DNA with an extraordinarily low fidelity (23,25,47). In yeast, Pol expression is induced by UV radiation (26,27). Thus, it is of particular interest to determine whether Pol is accessible to the DNA gap for repair synthesis during NER, especially when the amount of Pol is increased upon DNA damage. To do this, we performed repair synthesis assays in yeast pol2-16 mutant extracts. As presented above, deficient repair synthesis in this mutant extract could be complemented by purified yeast Pol (Fig. 7), suggesting that some DNA gaps were accumulated during NER in pol2-16 extracts. When we added excess amounts of purified yeast Pol (14-fold molar excess over DNA) during NER in pol2-16 mutant extracts, deficient repair synthesis could not be complemented (Fig. 8). These results suggest that Pol is not accessible to repair synthesis of NER in yeast cell-free extracts even when the polymerase is present in excess amount.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using biochemical analyses of in vitro repair in cell-free extracts, we show that the repair synthesis step of the yeast NER pathway requires both Pol
and Pol. Loss of either Pol or Pol activity reduced repair synthesis during NER in vitro. Nevertheless, residual repair synthesis was present, probably due to functional Pol in the Pol mutant extract or functional Pol in the Pol mutant extract. It is unlikely that the residual repair synthesis activity was derived from partial inactivation of Pol or Pol in the mutant extracts, because such residual activity was also observed even when the catalytic domain of Pol was deleted. Therefore, efficient NER in yeast cell-free extracts requires the participation of both Pol and Pol at the repair synthesis step. It is not clear mechanistically how the presence of both polymerases would mediate efficient repair synthesis of NER. One possibility is that both polymerases may be parts of a large protein complex for repair synthesis during NER. Loss of one polymerase may destabilize and/or reduce the activity of the repair synthesis complex. Alternatively, each repair synthesis complex may contain only one of the two polymerases. The presence of mutant Pol or Pol may form an inactive complex that competes for the functional complex at the repair synthesis step.
Our results indicate that during NER repair synthesis can occur without Pol or Pol, although less efficiently. These results are consistent with earlier reports that either Pol or Pol alone can carry out repair synthesis in an in vitro system reconstituted from purified human NER proteins (11,12). Overlapping or partially overlapping functions of Pol and Pol in repair synthesis of NER provide an explanation that the pol3-1 (cdc2-1), pol2-18 and pol2-16 mutant cells are not significantly sensitive to UV radiation (20,31) (X.Wu and Z.Wang, unpublished results). With significant repair synthesis activity still present in the mutant cells, it is expected that UV damage can be repaired by NER, even if it may take somewhat longer to do so. It is noted that some pol3 mutant alleles lead to cellular sensitivity to a DNA damaging agent, such as methyl methanesulfonate (MMS) (48). Since post-replication repair requires Pol (49), it is likely that the sensitivity is a consequence of defective post-replication repair rather than defective NER or BER.
Mammalian Polß plays a major role in BER (42). However, our in vitro results indicate that yeast Polß is not required for BER, regardless of whether the repair is initiated by a glycosylase with or without associated AP lyase. Polß is also not required for NER. Consistent with this conclusion, yeast cells without Polß are not sensitive to UV radiation or MMS (32,44). In fact, yeast Polß is expressed at a very low level during vegetative growth (32). Its expression, however, is stimulated during meiosis (32). Recently, Wilson and Lieber (50) showed that yeast Polß is involved in non-homologous DNA end joining, a special mode of DNA recombination. Therefore, the BER function of Polß appears to be acquired later during the evolution of higher eukaryotes. The repair synthesis step of BER in yeast is catalyzed by Pol (22). During yeast BER of uracil-containing DNA, the 5' deoxyribose phosphate moiety resulted from Apn1 incision is removed by the nuclease activities of Rad27 (51). The resulting gapped DNA can then be filled in by Pol and the nick is ligated by DNA ligase I (Cdc9 protein) (10). In higher eukaryotes, the Rad27 homolog is known as FEN1 and DNase IV (52,53), and Pol–Fen1 combination for BER is also reported (54,55). The Pol–Fen1 combination for BER may also occur in higher eukaryotes (42,56). Unlike yeast, however, this repair mechanism is thought to play only a minor role in human BER (42,54,56).
Recently, it was demonstrated that the yeast RAD30 gene codes for DNA Pol (26,27,39,57). Originally, Pol was identified as an error-free lesion bypass polymerase opposite TT dimers (39,58). Later, in vitro bypass of other DNA lesions by Pol was also demonstrated (35,59,60). Surprisingly, Pol copies DNA from undamaged templates with an extraordinarily low fidelity (23,25). For cells to maintain genomic stability, mechanisms must exist to exclude Pol from DNA replication. One mechanism is to regulate its expression. Indeed, transcription of yeast Pol is UV-inducible (26,27). In this study, we asked whether Pol is also excluded from NER in yeast. We found that excess amounts of purified yeast Pol were unable to complement deficient repair synthesis in pol2-16 mutant extracts. This result suggests that Pol cannot participate in repair synthesis during NER. Even purified Pol could not complement deficient repair synthesis in pol2-18 and pol2-16 mutant extracts, and purified Pol could not complement deficient repair synthesis in pol3-1 mutant extracts. Thus, it appears that following damage excision the resulting DNA gap is not exposed. Participation of Pol and Pol in repair synthesis of NER is likely to involve a recruitment mechanism, which may be coordinated with an earlier reaction in the NER pathway. This process is probably carried out through protein–protein interactions. Possible interactions include Rad2–PCNA, PCNA–Pol and PCNA–DNA ligase I, which have been reported (29,30,61). Thus, NER is most likely operated by protein complexes from damage recognition to DNA ligation in a coordinated manner.
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ACKNOWLEDGEMENTS |
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We thank Akio Sugino for providing yeast strains YHA302 and 488 and purified yeast DNA Pol
and Pol, David Hinkle for purified yeast DNA Pol, Christopher Lawrence for yeast strains CL1265-7C and AMY32, Judith Campbell for yeast strains TC102 and SK-2-1ß, and Curt Wittenberg for yeast strains CWY231 and TAY237. These studies were supported by a research grant CA67978 from NIH. D.G. is a recipient of the Kentucky Opportunity Fellowship.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 859 323 5784; Fax: +1 859 323 1059; Email: zwang{at}pop.uky.edu
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