Impairment of Proliferating Cell Nuclear Antigen-dependent Apurinic/Apyrimidinic Site Repair on Linear DNA*

Siham BiadeDagger , Robert W. Sobol§, Samuel H. Wilson§, and Yoshihiro MatsumotoDagger

From the Dagger  Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 and the § National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Repair of apurinic/apyrimidinic (AP) sites by mammalian cell extracts was compared using circular and linear DNA substrates. Extracts prepared from DNA polymerase beta  (polbeta )-proficient mouse fibroblasts repaired AP sites on both circular and linear DNA. However, extracts from the isogenic polbeta -knockout cells repaired AP sites on circular DNA but not efficiently on linear DNA. The circularity-dependent repair by the polbeta -knockout cell extract was completely inhibited by anti-proliferating cell nuclear antigen (PCNA) antibody but fully restored by addition of purified PCNA. Pretreatment of the linear DNA with AP endonuclease did not improve repair, indicating that impairment of AP site repair on linear DNA by polbeta -knockout cell extracts is not due to inefficiency of damage incision but rather to deficiency at the subsequent steps. These results indicate that AP sites can be repaired on circular DNA by the PCNA-dependent pathway in addition to the polbeta -dependent pathway and that the PCNA-dependent repair mechanism is poorly functional on linear DNA in vitro.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Base excision repair is a major mechanism by which cells correct bases with small modifications, AP1 sites, and single-strand breaks (1). These types of DNA damage are spontaneously generated through normal cellular metabolism and also are caused by exogenous agents such as ionizing radiation and alkylating and oxidizing agents. Thus, they belong to a group of the most abundant lesions in living cells. A typical process for base excision repair consists of five sequential reactions (2): (i) the modified base is removed by a specific DNA-N-glycosylase; (ii) the resultant AP site is incised at its 5' side by an AP endonuclease; (iii) a deoxyribose phosphate (dRP) is excised from the 5'-incised site; (iv) the gap is filled by DNA synthesis; and (v) the DNA strand is sealed by ligation. Several protein factors responsible for some steps in this process have been identified, including a group of DNA-N-glycosylases and AP endonucleases. Regarding the DNA synthesis step, however, it has not yet been established which enzymes are responsible for this step. Higher eukaryotes have five classes of DNA polymerases in nuclei, alpha , beta , delta , epsilon , and zeta  (3). Among them, polbeta has long been considered to be involved in base excision repair. Two recent observations strongly support this implication. First, a mouse fibroblast cell line in which both alleles of the polbeta gene are disrupted exhibits hypersensitivity to alkylating agents (4). Second, polbeta has beside its DNA polymerase activity, another enzymatic activity for the excision of dRP residues, an indispensable step for base excision repair (5). On the other hand, two studies using in vitro repair systems demonstrated that base excision repair can proceed by another pathway that requires PCNA as an essential factor (6, 7).

PCNA plays an essential role in DNA replication (8, 9) and in nucleotide excision repair (10, 11), and it has been shown recently to be involved in mismatch repair (12). It is a protein of approximately 29 kDa that forms a homotrimer with a torus structure. Double-stranded DNA can pass through the inside cavity of the PCNA trimer. Such loading of PCNA onto DNA is facilitated by the replication factor C (RF-C) in an ATP-dependent manner, resulting in a PCNA/RF-C complex known as the PCNA clamp (for review, see Ref. 13). The formation of the PCNA clamp is a prerequisite to efficient DNA synthesis by DNA polymerase delta  (poldelta ) or polymerase epsilon  (polepsilon ). Because PCNA stimulates the activities of poldelta and polepsilon but not polymerase alpha  or polymerase beta , the alternative pathway for base excision repair should use poldelta and/or polepsilon at its DNA synthesis step. In contrast, several studies which also used in vitro repair systems indicated that polbeta was the exclusive DNA polymerase responsible for base excision repair (4, 14, 15). We noticed that the studies demonstrating the PCNA-dependent pathway used circular DNA substrates, whereas linear DNA substrates were used in the studies supporting the polbeta -dependent pathway in exclusion of the PCNA-dependent pathway. Podust et al. (16) reported that PCNA could be loaded more stably on circular DNA than on linear DNA in vitro. Unstable loading of the PCNA onto linear DNA is due to the falling off of the PCNA clamp from linear DNA ends after sliding along it. If this property of PCNA-DNA binding applies to base excision repair, linear DNA substrates may not support the PCNA-dependent pathway. To test this possibility, we compared the repair efficiencies on circular and linear DNA side by side using extracts prepared from wild-type and polbeta -knockout mouse cells. We show here that DNA substrates that do not have free ends are preferable to detect PCNA-mediated in vitro repair.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cell Lines-- The wild-type mouse embryonic fibroblast cell line and the matched littermate polbeta -knockout cell line (4) were maintained in Glutamax I medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Atlanta Biologicals), 100 units/ml penicillin, 100 µg/ml streptomycin, and 80 µg/ml hygromycin B (Sigma). The cells were cultured in monolayer in a humidified incubator with 10% CO2 at 34 °C. CHO-K1 and its double-strand break repair-deficient derivative, xrs-5, were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine in a humidified incubator with 5% CO2 at 37 °C.

Preparation of Cell Extracts-- Cells were cultured in 175 cm2 flasks and incubated overnight to reach mid-exponential growth phase. The cells were then washed three times with ice-cold phosphate-buffered saline and resuspended at 106 cells/20 µl in Buffer I (10 mM Tris-Cl, pH 7.8, and 200 mM KCl). After the addition of an equal volume of Buffer II (10 mM Tris-Cl, pH 7.8, 200 mM KCl, 2 mM EDTA, 40% glycerol, 0.2% Nonidet P-40, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin), the cell suspension was rocked at 4 °C for 1 h and then centrifuged at 16,000 × g for 10 min. The supernatant was recovered and stored in small aliquots at -80 °C.

Preparation of DNA Substrates Containing an AP Site-- Covalently closed circular DNA (cccDNA) which carried either a uridine or a synthetic AP site analog (3-hydroxy-2-hydroxymethyltetrahydrofuran) was prepared as described previously (6). In prelabeled cccDNA, 32P was incorporated at the position several nucleotides away from the lesion. Linear DNA was prepared by digestion of the cccDNA with PvuII, which generated a 382-base pair fragment carrying the AP site in its middle position and a 2.8-kilobase fragment with no AP site. For natural AP site repair assays, the DNA containing a uridine residue was treated with uracil-DNA glycosylase (Perkin-Elmer) immediately before the repair reaction as described previously (6) to create a natural AP site.

DNA Repair Assay-- A standard repair reaction was carried out at 25 °C with 10 ng of a DNA substrate (200-1000 cpm, 5 fmol AP sites) and 10 µg of the cell extract protein in a 20-µl reaction mixture (20 mM HEPES-KOH, pH 7.5, 10 mM MgCl2, 135 mM KCl, 1 mM dithiothreitol, 2 mM ATP, 20 µM each of four dNTPs, 2 mM NAD, 40 mM phosphocreatine, and 1 unit of creatine phosphokinase). When indicated, repair factors were added to the cell extracts immediately before the reaction was started. In experiments for neutralizing PCNA, the antibody AK specific for PCNA (37.5 µg/ml) (generously provided by Dr. M. Miura, Tokyo Medical and Dental University) was incubated with the cell extracts on ice for 20 min before the repair reaction. At indicated times the reaction was stopped by the addition of sodium dodecyl sulfate to a final concentration of 0.4% and 2 µg of proteinase K followed by a 30-min incubation at 37 °C. The DNA was recovered by phenol:chloroform extraction and ethanol precipitation and digested with AP endonuclease and PvuII (for cccDNA only). The DNA was then subjected to electrophoresis on an 8 M urea-containing 6% polyacrylamide gel. Repaired and unrepaired DNA fragments were detected by autoradiography, and the amounts of radioactivities were quantified with a Fuji BAS 1000 phosphorimager.

Detection of Uncut AP Sites-- After the repair reaction, DNA was digested with KpnI and PvuII (for cccDNA only) for 1 h at 37 °C and subjected to electrophoresis on an 8 M urea-containing 6% polyacrylamide gel. KpnI can cleave only the repaired DNA substrate but not the unrepaired DNA substrate, which has an AP site in the KpnI restriction site. The 382-nucleotide fragment that carried an intact AP site and the 131-nucleotide and 129-nucleotide fragments which were generated by the AP endonuclease digestion during the repair reaction or by the KpnI digestion of the repaired product were detected by autoradiography, and the amounts of radioactivities were quantified with a Fuji BAS 1000 phosphorimager.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Procedures for Cell Extract Preparation-- Most of the studies dealing with in vitro DNA repair assays with mammalian cultured cells utilize extracts prepared by the procedures described by Manley et al. (17) with some modifications. This technique is time consuming and requires large amounts of cells. We prepared extracts from detergent-lysed cells by the procedures described by Tanaka et al. (18) with a few modifications (see under "Experimental Procedures" for details). These procedures allowed us to obtain from a million cells an amount of cell extract sufficient for 30 repair reactions. In a preliminary experiment, we compared the repair activity of cell extracts prepared by this method with that of the extracts prepared as described by Manley et al. (17). These extracts were made from mouse polbeta -knockout cells and their isogenic wild-type cells. Repair assays were conducted on AP site-containing circular DNA. AP sites are common intermediate products generated during base excision repair and are efficiently repaired by Xenopus laevis ovarian extracts (6) and mammalian cell extracts (7). It has been demonstrated with a reconstituted repair system using X. laevis proteins that a synthetic AP site analog, 3-hydroxy-2-hydroxymethyltetrahydrofuran, is mostly repaired by the PCNA-dependent pathway, whereas the natural AP site is repaired by both the polbeta -dependent pathway and the PCNA-dependent pathway (6). Therefore, we also compared repair of these two types of lesions. As shown in Table I, both cell extracts prepared from wild-type and polbeta -knockout cells were able to repair AP sites, although less efficiently by the latter. This result indicates that AP sites were repaired, at least in part, by a mechanism independent of polbeta in these mouse cell extracts. In each case, the extract prepared by the method of Tanaka et al. provided higher repair activity than the extract prepared by the method of Manley et al. Therefore, the subsequent experiments in this study were carried out using cell extracts prepared by the procedure based on the detergent cell lysis.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Comparison of the repair activity of cell extracts prepared by two different methods
Extracts were prepared from indicated cells by the method of Tanaka et al. (18) (I) or the method of Manley et al. (17) (II). Repair reactions with circular DNA carrying the indicated lesions were carried out for 30 min at 25 °C as described under "Experimental Procedures."

Influence of DNA Structure on AP Site Repair-- To evaluate more accurately the repair activity of each extract, a time course of the repair was measured. On circular DNA, the synthetic AP site analog was repaired at the same rate by both wild-type and polbeta -knockout cell extracts (Fig. 1A). In contrast, the natural AP site was more efficiently repaired by wild-type cell extracts (Fig. 1B), indicating that a portion of repair in the wild-type cell extract was mediated by a polbeta -dependent pathway. The initial rate of natural AP site repair by the polbeta -knockout cell extract was similar to that observed for the synthetic AP site repair by both extracts. Thus, the difference of the natural AP site repair between the two extracts could be due to preferential repair of the natural AP sites over the synthetic AP sites by the polbeta -dependent mechanism. As previously reported, polbeta can excise a dRP residue from the 5'-incised AP site by its own dRP lyase activity (5). However, polbeta cannot excise a dRP residue from the synthetic AP site analog, because this lesion is resistant to beta -elimination. It was observed that synthetic AP sites were not efficiently repaired by the polbeta -dependent pathway (6). On linear DNA, however, only the wild-type cell extracts were able to repair efficiently the natural AP site and to a lesser extent the synthetic AP site analog (Fig. 1, C and D). The complete lack of DNA repair with the polbeta -knockout cell extracts on linear DNA indicates that only the polbeta -dependent pathway is functional on this DNA. Thus, the alternative pathway cannot repair either type of AP sites on linear DNA. The effect of DNA linearization on AP site repair appeared to have an inverse correlation with the length of the linear DNA. Although the PvuII-digested DNA (382 base pairs) was not repaired at all by polbeta -knockout cell extracts (Fig. 1), the XmnI-linearized DNA (3.2 kilobases) was repaired by the same extracts with approximately 50% efficiency of that of circular DNA (data not shown).


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 1.   Influence of DNA structure on AP site repair. Repair reactions were carried out by incubating circular or linear DNA carrying either a natural AP site or a synthetic AP site analog with the extract from wild-type mouse fibroblasts (wt) or polbeta -knockout (polbeta -/-) cells for 0, 15, 30, 60, and 90 min as described under "Experimental Procedures." A, repair of the synthetic AP site on circular DNA. B, repair of the natural AP site on circular DNA. C, repair of the synthetic AP site analog on linear DNA. D, repair of the natural AP site on linear DNA.

According to the previously reported studies (6, 7), this alternative pathway for base excision repair should proceed through the PCNA-dependent DNA synthesis. To test whether this is the case in the present repair system with mouse cell extracts, we used an anti-PCNA antibody (AK) known to have a neutralizing activity for the PCNA-dependent DNA synthesis (19). When incubated for 20 min with the cell extracts prior to the reaction, this antibody completely inhibited the repair reaction carried out by polbeta -knockout cell extracts on circular DNA (Fig. 2). Moreover, this repair was fully restored by addition of 500 ng of PCNA to the reaction. These results clearly indicated that PCNA is required for the alternative pathway in the polbeta -knockout cell extracts. The repair of the synthetic AP site analog by the wild-type cell extracts was only partially inhibited. This is because this synthetic lesion can be repaired by the polbeta pathway, although in a less efficient manner. The synthetic AP site analog cannot be excised by polbeta , because this lesion is refractory to beta -elimination (5). However, it is reported that FEN1 (also known as DNaseIV) can excise modified AP sites which cannot be removed by beta -elimination (20). Therefore, the excision step of the synthetic AP sites was probably catalyzed by a FEN1-like enzyme present in the cell extracts. Once this step is accomplished, polbeta should ensure the completion of the repair. We indeed observed that addition of FEN1 to the reconstituted system for the polbeta -dependent AP site repair rescued the repair of synthetic AP sites.2 A similar inefficient repair of the synthetic AP site analog by the polbeta -dependent pathway is also shown in Fig. 1C (compare with Fig. 1D). The AK antibody did not inhibit at all the natural AP site repair by the wild-type cell extracts, because this repair can be catalyzed by the polbeta -dependent pathway without restriction. Taken together, the PCNA-dependent AP site repair can proceed in vitro on circular DNA but not efficiently on linear DNA.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibition of repair by anti-PCNA antibody. Wild-type cell extracts and polbeta -/- cell extracts were preincubated with AK antibody when indicated and used for repair reactions of circular DNA carrying either a synthetic AP site analog (A) or a natural AP site (B) as described under "Experimental Procedures." When indicated, 500 ng of PCNA were added immediately before the repair reaction. The repair reactions were carried out for 1 h.

Effect of Additional Repair Factors on AP Site Repair-- To confirm further the differential effect of DNA structure between the polbeta -dependent pathway and the PCNA-dependent pathway, we stimulated either one of the two pathways by adding purified polbeta or PCNA to the repair reactions with the cell extracts. When we added 50 ng of rat polbeta , the AP sites were mostly repaired in any combination of the cell extracts and the DNA substrates (Fig. 3), indicating that the polbeta -dependent pathway was fully functional on both circular and linear DNA substrates. On the other hand, the addition of 50 ng of mouse PCNA increased the repair efficiency on the circular DNA but did not rescue the repair on the linear DNA (Fig. 4). Thus, the stimulating effect of additional PCNA on AP site repair was limited to circular substrates. The increase in AP site repair by additional PCNA was relatively small compared with that by additional polbeta . This is probably because PCNA may not be the only limiting factor for this repair pathway. Poldelta , polepsilon , and RF-C may also be limiting, as such large factors may not be efficiently extracted from the nuclei during our cell extract preparation procedures.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of additional polbeta on DNA repair. Repair reactions were carried out for 1 h without (open bars) or with 50 ng of rat polbeta (hatched bars) in addition to the indicated extract as described under "Experimental Procedures."


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of additional PCNA on AP site repair. Repair reactions were carried out for 30 min without (open bars) or with 50 ng mouse PCNA (hatched bars) as described under "Experimental Procedures" in addition to the indicated extract.

Inhibition of Base Excision Repair in Linear DNA Is Not Due to Ku86 Protein-- Recently, Calsou et al. (21) reported that nucleotide excision repair was inhibited by double-strand breaks in a cis-acting manner, and that this inhibition could be due to the blocking by Ku proteins of the damage-specific incision. Ku proteins which can bind as a heterodimer of Ku70 and Ku86 to double-strand breaks are required for double-strand break DNA repair and V(D)J recombination (22, 23). Calsou et al. (21) observed that linearization of DNA substrates reduced the nucleotide excision repair activity to 50% in the extracts from Ku-proficient CHO-K1 cells but did not affect the repair in the extracts from Ku86-deficient xrs-6 cells. We investigated a possible involvement of Ku proteins in the AP site repair process on linear DNA. Cell extracts were prepared from Ku86-deficient xrs-5 cells and the parental Ku-proficient CHO-K1 cells, and their repair activities were compared on linear and circular DNA carrying either a synthetic or a natural AP site. Because the CHO cell lines were proficient in polbeta activity, natural AP sites on linear DNA were repaired in the extracts from these cells (Fig. 5). The repair of the synthetic AP site analog was, in contrast, relatively inefficient on linear DNA. This repair impairment was observed at similar levels in both CHO-K1 and xrs-5 cell extracts. Furthermore, the AP site on linear DNA was incised at the same rate by both cell extracts. The repair reactions with CHO-K1 and xrs-5 cell extracts left 6 and 12% of AP sites on linear DNA uncut (Table II), indicating that 94 and 88%, respectively, of the DNA substrate was incised by AP endonucleases contained in these cell extracts. On circular DNA, the incision rate was higher than 95% for both cell extracts. This result shows that the Ku86 protein did not inhibit the incision step on either circular or linear DNA. We also examined AP site repair of DNA pretreated with AP endonuclease. As shown in Fig. 6, preincision of AP sites did not improve the repair of linear DNA by the polbeta -knockout cell extracts, indicating that the impairment of PCNA-dependent pathway on linear DNA is not due to inefficient incision of AP sites. These results ruled out the possible role of the Ku86 protein in the inhibition of base excision repair on linear DNA in our system.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of Ku86 protein on DNA repair. A, repair with CHO-K1 cell extracts. B, repair with xrs-5 cell extracts. Repair reactions on circular DNA (open symbols) or linear DNA (closed symbols) carrying either a natural AP site (circles) or a synthetic AP site analog (squares) were carried out for the indicated times as described under "Experimental Procedures."

                              
View this table:
[in this window]
[in a new window]
 
Table II
Detection of intact AP sites remaining after repair reaction


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of AP endonuclease pretreatment on DNA repair. Repair reactions were carried out for 60 min with polbeta -/- cell extracts on the untreated DNA (open bars) or on the DNA treated with AP endonuclease (hatched bars) as described under "Experimental Procedures."

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this report, we have demonstrated that AP sites can be repaired by the polbeta -dependent pathway and an alternative polbeta -independent pathway in extracts prepared from mouse fibroblast-derived cells. The alternative pathway uses PCNA as one of its essential factors. This PCNA-dependent pathway was functional on circular DNA but not on linear DNA in vitro, whereas the polbeta -dependent repair reaction proceeded efficiently on both circular and linear substrates. These results resolve the controversy raised by the studies previously reported (4, 6, 7, 14, 15). In addition, it is clear that circular DNA should be used as a substrate for in vitro repair assays when PCNA may be involved in the reaction. Recently, Klungland and Lindahl (20) reported that PCNA enhanced the polbeta -dependent repair of modified AP sites by stimulating the FEN-1 activity. They also observed that polbeta -neutralizing antibodies decreased this AP site repair to 5%, claiming polbeta as the major enzyme for DNA synthesis in base excision repair. Because they used a double-stranded oligonucleotide as a substrate, it is likely that their results may underestimate the contribution of PCNA-dependent DNA polymerases poldelta /polepsilon to base excision repair. The study performed with Xenopus purified enzymes demonstrated that poldelta repaired efficiently AP sites on circular DNA (6). The data reported here with polbeta -knockout cell extracts also suggest that DNA synthesis for the PCNA-dependent repair should be attributed to poldelta or polepsilon .

One explanation for the mechanism of in vitro repair inhibition on linear DNA was provided by Calsou et al. (21) for nucleotide excision repair in which the damage-specific incision may be blocked by Ku proteins through their binding to the DNA ends. However, our results with xrs-5 cell extracts and those obtained with the DNA preincised with AP endonuclease indicate that this was not the case for the impairment of AP site repair that we observed on linear DNA. Although the PCNA-dependent base excision repair and nucleotide excision repair use a PCNA-dependent DNA polymerase, either delta  or epsilon , their incision mechanisms of damaged sites are distinct from each other. Nucleotide excision repair requires at least 16 polypeptides for incision of damaged sites (24). This incision step seems to be rate-limiting in the repair reaction with mammalian cell extracts rather than the DNA synthesis step (25). In contrast, AP endonuclease is sufficient by itself for incision of AP sites in base excision repair.

Inability to repair AP sites on linear DNA by the PCNA-dependent pathway results most likely from the property of the PCNA clamp on DNA. Podust et al. (16) demonstrated that the RF-C·PCNA complex assembled on gapped circular DNA was easily lost once the DNA was linearized. Consequently, poldelta with PCNA and RF-C cannot carry out DNA synthesis on linear DNA as efficiently as on circular gapped DNA. Studies with X. laevis reconstituted systems suggest that RF-C is required in the PCNA-dependent AP site repair (6). The efficient utilization of circular DNA as a template for PCNA-dependent reactions seems to be due to its structural character, for that circular DNA does not have free ends. In fact, Yao et al. (26) reported that blocking of linear DNA ends with a specific DNA-binding protein reduces the dissociation of the PCNA clamp from DNA. This situation resembles the DNA repair reaction in living cells. There is a model for in vivo repair in which DNA repair may be associated with rearrangements in chromatin structure including dissociation of nucleosomes around damaged sites (27). Therefore, a free slide of the PCNA clamp could be limited within the unfolded region of the damaged chromosomal DNA.

    ACKNOWLEDGEMENTS

We thank M. Miura for the AK antibody and S. W. Johnson, T. C. Hamilton, and A. T. Yeung for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA63154 and CA 06927 and an appropriation from the Commonwealth of Pennsylvania.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.

To whom correspondence should be addressed: Dept. of Radiation Oncology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-5272; Fax: 215-728-4333; E-mail: y_matsumoto{at}fccc.edu.

1 The abbreviations used are: AP, apurinic/apyrimidinic; polbeta , DNA polymerase beta ; poldelta , DNA polymerase delta ; polepsilon , DNA polymerase epsilon ; PCNA, proliferating cell nuclear antigen; dRP, deoxyribose phosphate; cccDNA, covalently closed circular DNA; RF-C, replication factor C.

2 K. Kim, S. Biade, and Y. Matsumoto, submitted for publication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Wallace, S. S. (1994) Int. J. Radiat. Biol. 66, 579-589[Medline] [Order article via Infotrieve]
  2. Lindhal, T. (1995) J. Cell Sci. 19, 73-77
  3. Wood, R. D., and Shivji, M. K. K. (1997) Carcinogenesis 18, 605-610[Abstract]
  4. Sobol, R. W., Horton, J. K., Kuhn, R., Gu, H., Singhal, R. K., Prasad, R., Rajewsky, K., Wilson, S. H. (1996) Nature 379, 183-186[CrossRef][Medline] [Order article via Infotrieve]
  5. Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702[Medline] [Order article via Infotrieve]
  6. Matsumoto, Y., Kim, K., and Bogenhagen, D. F. (1994) Mol. Cell. Biol. 14, 6187-6197[Abstract]
  7. Frosina, G., Fortini, P., Rossi, O., Carrozzino, F., Raspaglio, G., Cox, L. S., Lane, D. P., Abbondandolo, A., Dogliotti, E. (1996) J. Biol. Chem. 271, 9573-9578[Abstract/Free Full Text]
  8. Prelich, G., Kostura, M., Marshak, D. R., Mathews, M. B., Stillman, B. (1987) Nature 326, 471-475[CrossRef][Medline] [Order article via Infotrieve]
  9. Bravo, R., Frank, R., Blundell, P. A., Macdonald-Bravo, H. (1987) Nature 326, 515-517[CrossRef][Medline] [Order article via Infotrieve]
  10. Shivji, M. K. K., Kenny, M. K., and Wood, R. D. (1992) Cell 69, 367-374[Medline] [Order article via Infotrieve]
  11. Nichols, A. F., and Sancar, A. (1992) Nucleic Acids Res. 20, 2441-2446[Abstract]
  12. Umar, A., Buermeyer, A. B., Simon, J. A., Thomas, D. C., Clark, A. B., Liskay, R. M., Kunkel, T. A. (1996) Cell 87, 65-73[Medline] [Order article via Infotrieve]
  13. Kelman, Z. (1997) Oncogene 14, 629-640[CrossRef][Medline] [Order article via Infotrieve]
  14. Dianov, G., Price, A., and Lindahl, T. (1992) Mol. Cell. Biol. 12, 1605-1612[Abstract]
  15. Wiebauer, K., and Jiricny, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5842-5845[Abstract]
  16. Podust, L. M., Podust, V. N., Floth, C., and Hubscher, U. (1994) Nucleic Acids Res. 22, 2970-2975[Abstract]
  17. Manley, J. L., Fire, A., Samuels, M., and Sharp, P. A. (1983) Methods Enzymol. 101, 568-582[Medline] [Order article via Infotrieve]
  18. Tanaka, M., Lai, J. S., and Herr, W. (1992) Cell 68, 755-767[Medline] [Order article via Infotrieve]
  19. Cheng-Keat, T., Sullivan, K., Li, X., Tan, E. M., Downey, K. M., So, A. G. (1987) Nucleic Acids Res. 22, 9299-9308
  20. Klungland, A., and Lindahl, T. (1997) EMBO J. 16, 3341-3348[Abstract/Free Full Text]
  21. Calsou, P., Frit, P., and Salles, B. (1996) J. Biol. Chem. 271, 27601-27607[Abstract/Free Full Text]
  22. Boubnov, N. V., Hall, K. T., Wills, Z., Lee, S. E., He, D. M., Benjamin, D. M., Pulaski, C. R., Band, H., Reeves, W., Hendrickson, E. A., Weaver, D. T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 890-894[Abstract]
  23. Taccioli, G. E., Gottlieb, T. M., Blunt, T., Priestley, A., Demengeot, J., Mizuta, R., Lehmann, A. R., Alt, F. W., Jackson, S. P., Jeggo, P. A. (1994) Science 265, 1442-1445[Medline] [Order article via Infotrieve]
  24. Mu, D., Park, C-H., Matsunaga, T., Hsu, D. S., Reardon, J. T., Sancar, A. (1995) J. Biol. Chem. 270, 2415-2418[Abstract/Free Full Text]
  25. Hansson, J., Grossman, L., Lindhal, T., and Wood, R. D. (1990) Nucleic Acids Res. 18, 35-40[Abstract]
  26. Yao, N., Turner, J., Kelman, Z., Stukenberg, P. T., Dean, F., Shechter, D., Pan, Z-Q, Hurwitz, J., O'Donnell, M. (1996) Genes Cell 1, 101-113 [Abstract/Free Full Text]
  27. Smerdon, M. J. (1991) Curr. Opin. Cell Biol. 3, 422-428[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.