COMMUNICATION
Role of DNA Polymerase beta  in the Excision Step of Long Patch Mammalian Base Excision Repair*

Grigory L. DianovDagger §, Rajendra Prasad, Samuel H. Wilson, and Vilhelm A. BohrDagger

From the Dagger  NIA, National Institutes of Health, Baltimore, Maryland 21224 and the  NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The two base excision repair (BER) subpathways in mammalian cells are characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" BER pathway and the "long patch" (several nucleotides incorporated) BER pathway. Both of these subpathways involve excision of a damaged base and/or nearby nucleotides and DNA synthesis to fill the excision gap. Whereas DNA polymerase beta  (pol beta ) is known to participate in the single-nucleotide BER pathway, the identity of polymerases involved in long patch BER has remained unclear. By analyzing products of long patch excision generated during BER of a uracil-containing DNA substrate in mammalian cell extracts we find that long patch excision depends on pol beta . We show that the excision of the characteristic 5'-deoxyribose phosphate containing oligonucleotide (dRP-oligo) is deficient in extracts from pol beta  null cells and is rescued by addition of purified pol beta . Also, pol beta -neutralizing antibody inhibits release of the dRP-oligo in wild-type cell extracts, and the addition of pol beta  after inhibition with antibody completely restores the excision reaction. The results indicate that pol beta  plays an essential role in long patch BER by conducting strand displacement synthesis and controlling the size of the excised flap.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Base excision repair of uracil in DNA is initiated by uracil-DNA glycosylase, which removes uracil by cleavage of the base-sugar glycosidic bond (1). The AP site1 resulting from this DNA glycosylase activity is then processed by AP endonuclease, DNA polymerase beta  (pol beta ), and DNA ligase I (2, 3), resulting in the replacement of one nucleotide. Processing of the AP site can also be accomplished by a different subpathway of BER, resulting in a longer DNA repair patch of several nucleotides (4, 5). A satisfactory understanding of the enzymes participating in this "long patch" BER and the mechanisms involved has not been achieved. It was shown that long patch BER reconstituted with partially purified components depended on PCNA (4) and that long patch BER in cell extract is sensitive to PCNA antibody (5). It was suggested that DNA polymerases delta  or epsilon  are involved in the gap-filling step during long patch BER (4, 5) because these enzymes are known to be stimulated by PCNA and these polymerases are proficient in reconstituted long patch BER systems (6, 7). Alternatively, if the role of PCNA in long patch BER is limited to the stimulation of FEN1 cleavage of a flap DNA substrate (8-10), other polymerases may also be considered as participants in long patch BER. There are observations suggesting that pol beta  can operate during long patch BER. Both DNA polymerases beta  and delta  can function in long patch BER reconstituted with purified proteins (7), and it was demonstrated that cell extract-mediated long patch BER is inhibited by antibody to pol beta  (7). Also, pol beta  null cell extract does not repair a reduced abasic site in a linear DNA substrate that is repaired through long patch BER in pol beta -containing wild-type cell extract (11). Further, the absolute amount of long patch BER activity (substrate repaired per mg of extract protein) in the pol beta  null cell extract is less than that in the isogenic wild-type cell extract.2 Taken together, these results suggest that pol beta , in addition to its role in single-nucleotide patch repair, could play an important but yet unknown role in long patch BER. In light of this background information, we directly addressed the role of pol beta  in the excision process of long patch BER performed by mammalian cell extracts.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and Extracts-- Normal human lymphoid cell line AG9387 was obtained from the Human Genetic Mutant Cell Repository (Coriell Institute, Camden, NJ). Cells were grown in medium recommended by the supplier. The DNA pol beta -knockout mouse fibroblasts and the isogenic wild type cell lines were grown as described (12). Whole cell extracts were prepared from 3-5 g of cells by the method of Manley et al. (13) and dialyzed overnight against buffer containing 25 mM Hepes-KOH, pH 7.9, 2 mM dithiothreitol, 12 mM MgCl2, 0.1 mM EDTA, 17% glycerol, and 0.1 M KCl. Extracts were aliquoted and stored at -80 °C.

Proteins and Antibodies-- Human DNA pol beta  was purified as described (14). Polyclonal antibodies against human pol beta  were raised in rabbit and were affinity-purified on a pol beta -Sepharose column.

Construction of Closed Circular M13 DNA Containing a Single Uracil Residue-- Double-stranded closed circular DNA containing single uracil (U-DNA) was constructed as described (15) by priming single-stranded M13 DNA with the 5'-labeled oligonucleotide 32pUCGGCCGATCAAGCTTATTGGGTACCG for internally labeled uracil-containing substrate and 32pCCGGCCGATCAAGCTTATTGGGTACCG for the control DNA substrate.

Excision Assay-- Standard 10-µl reactions contained 50-100 ng of internally labeled U-DNA, 45 mM Hepes-KOH (pH 7.8), 70 mM KCl, 7.5 mM MgCl2, 1 mM dithiothreitol, 0.4 mM EDTA, 2 mM ATP, 3.4% glycerol, 50 µM each of dGTP, dATP, TTP, dCTP, 1 µg of 30-mer single-stranded oligonucleotide (as a carrier), and 20 µg of a cell extract protein. DNA repair synthesis reactions were carried out at 32 °C for the indicated times. After the reaction, excision products were stabilized by the addition of 0.5 M NaBH4 to a final concentration of 0.1 M and incubated for 30 s on ice. Then 12 µl of formamide-dye solution was added, and the reaction products were separated by electrophoresis on a 20% denaturing polyacrylamide gel.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Excision Products Generated by Human Cell Extract-- To assess the role of pol beta  in long patch BER of uracil-DNA (U-DNA), we analyzed BER excision products formed in cell extracts. A closed circular plasmid DNA substrate containing a 32P-labeled phosphate group immediately 5' to a solitary uracil-guanine base pair (Fig. 1a) was prepared. To release the labeled phosphate from the substrate, enzymes in a cell extract must first excise uracil and then incise the phosphodiester bond 5' to the arising AP site. Next, the labeled phosphate is released either as the 5'-deoxyribose phosphate residue (dRP) or as dRP moiety attached to a short oligonucleotide produced after strand displacement and flap incision (dRP-oligo). Under our standard reaction conditions, >70% of the U-DNA substrate was consumed in 60 min (Fig. 1, a and b). In addition to release of a dRP, which migrated very quickly out of the gel, there was release of a predominant dRP-oligo product that almost co-migrated with a trinucleotide marker (Fig. 1b). A control DNA substrate with a normal cytosine-guanine base pair at the same position as the uracil-guanine lesion was not degraded (Fig. 1b, lane C).


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Fig. 1.   Long patch excision products generated during repair of U-DNA by human cell extract. a, U-DNA substrate containing a single-uracil residue and labeled phosphate group (in bold) 5' next to uracil is shown. b, 100 ng of internally labeled U-DNA was incubated with 20 µg of human whole cell extract at 32 °C for the indicated time period. Control reaction (lane C) containing 100 ng of internally labeled substrate DNA with a regular C:G base pair at the same position was incubated with 20 µg of human whole cell extract at 32 °C for 1 h. After the incubation, reaction products were reduced with sodium borohydride and analyzed by electrophoresis on 20% polyacrylamide gel. c, the 5'-deoxyribose phosphate oligonucleotide dRpCpGpG (lane 1), was generated as described in text and electrophoresed on 20% polyacrylamide gel next to the markers (lane 2).

Because oligonucleotides containing 5'-dRP may migrate differently in polyacrylamide gels than the oligonucleotide markers used, we engineered a marker with the same nucleotide sequence as the expected excision product and with the dRP residue at the 5'-end. First, we constructed an oligonucleotide duplex with a uracil residue in one strand 4 bases upstream of the 3'-end and with a labeled phosphate group next to it on the 5' side. This oligonucleotide duplex was then treated with bacterial uracil-DNA glycosylase and endonuclease IV. The combined action of these enzymes releases the 5'-deoxyribose phosphate-containing oligonucleotide pdRpCpGpG. This product was stabilized by reduction with NaBH4 and subjected to high resolution electrophoresis. The pdRpCpGpG molecule almost co-migrated with the 3-mer marker (Fig. 1c). We conclude that during long patch BER of the U-DNA the dRP residue can be excised with at least three nucleotides located immediately 3' to the damaged base.

Excision of dRP-oligo Depends on pol beta -- The use of pol beta -knockout mouse embryonic fibroblasts with a homozygous deletion in the pol beta  gene allowed us to test directly whether pol beta  is involved in excision steps of long patch BER. We found that wild-type mouse cell extract released dRP-oligo as a major excision product in long patch BER. This product was strongly reduced in the pol beta  null cell extract, and the size distribution of excision products was different (Fig. 2). To confirm that the observed reduction in dRP-oligo release was due to the absence of pol beta , the purified enzyme was added to cell extract prepared from pol beta  null cells (Fig. 3). As little as 2 ng of pol beta  could reconstitute dRP-oligo release to the level observed in wild-type cell extract (Fig. 3, compare lanes 4 and 6). The addition of 4 ng of pol beta  further stimulated dRP-oligo excision (Fig. 3, compare lanes 5 and 6).


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Fig. 2.   The release of dRP-oligo in cell extracts. 100 ng of internally labeled U-DNA was incubated with 20 µg of extract of the wild-type or pol beta  null cells at 32 °C for the indicated time period as described under "Materials and Methods." After the incubation, reaction products were reduced with sodium borohydride and analyzed by electrophoresis on 20% polyacrylamide gel.


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Fig. 3.   Reconstitution of dRP-oligo release in a pol beta  null cell extract with purified pol beta . Cell extract (20 µg of protein) derived from pol beta  null cells was preincubated on ice for 20 min with the indicated amounts of pol beta  protein before the addition of U-DNA substrate. Reactions were further incubated for 20 min at 32 °C and processed as described above. The release of dRP-oligo by wild-type (pol beta +) extract is shown in lane 6.

Additional evidence for a role of pol beta  in excision was obtained by using antibody specific to pol beta . The addition of antibody blocked the excision of dRP-oligo and resulted in an excision product pattern characteristic for the pol beta  null cell extract (Fig. 4, lanes 2-4). This excision deficiency in the presence of antibody was then corrected by addition of pol beta  to the antibody-containing reaction (Fig. 4, lane 5), indicating that the effect of antibody was highly specific and limited to blockage of pol beta  function. This antibody to pol beta  is known to inhibit the enzyme's DNA polymerase activity (16). Based on these experiments, we conclude that DNA repair synthesis performed by pol beta  is required for dRP-oligo excision in long patch BER.


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Fig. 4.   pol beta  dependence of dRP-oligo release in a wild-type cell extract. Cell extract (20 µg) derived from wild-type mouse cells was preincubated on ice for 20 min with indicated amounts of pol beta  polyclonal antibodies before the substrate U-DNA was added. Reactions were further incubated for 20 min at 32 °C. Lane 5 also contains 4 ng of pol beta  in addition to the pol beta  antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DNA Polymerases in Base Excision Repair-- Previous studies had shown that pol beta  is the major DNA polymerase for the single-nucleotide BER pathway (12) whereas pol delta /epsilon are thought to be involved in PCNA-dependent long patch BER (4, 5). Recent findings, however, suggest that pol beta  and pol delta  can substitute for each other in long patch BER reconstituted with purified proteins (7). The substitution of different polymerases in BER was also confirmed by the competence of pol beta  null cell extracts in the in vitro repair of both natural and reduced abasic sites in closed circular DNA (11, 17). Thus, the biochemical proficiency of pol beta  and other polymerases in both subpathways for base excision repair has been documented. Yet, the question remained as to the DNA polymerase of choice for the long patch BER subpathway and its precise role(s) in the sequential mechanism. In this report we present data demonstrating that pol beta  is the major DNA polymerase involved in long patch BER in mammalian cells. Several experimental approaches used in this study support this conclusion. First, the excision step of long patch BER is dependent upon the pol beta  status of the cell extract: long patch excision is reduced in pol beta -deficient cells but can be reconstituted by the addition of purified pol beta . Second, pol beta -neutralizing antibody inhibits long patch BER excision and especially release of the dRP-oligo product. These results were unexpected because pol beta  has not been proposed to participate in the long patch BER reaction. The striking homogeneity of the excision product size (i.e. the dRP-oligo) indicates that the proteins involved in excision may predetermine the length of the excised oligonucleotide, and pol beta  is a good candidate for this function. It was shown earlier that when a suitable substrate is provided, FEN1 is able to release the AP-site 5'-sugar phosphate as part of an oligonucleotide but does not favor any particular flap size (18). However, as we have demonstrated here in the presence of pol beta , the excision is almost strictly limited to release of the dRP-oligo, and in pol beta -deficient cell extract this particular excision product was significantly reduced.

The Role of pol beta  in Coordination of BER-- It appears that different steps of the BER reaction are coordinated and directed by multiple interactions between the participating proteins (3). For example, after removal of the T base, from the G-T mismatch, G-T-DNA glycosylase remains bound to the AP site and must be displaced by AP endonuclease (19). Further, AP endonuclease was shown to interact with pol beta , and this interaction stimulates the pol beta  lyase repair reaction (20). After this step in the repair process pol beta  appears to play a central role in determining the subpathway: removal of 5'-sugar phosphate by pol beta  (21, 22) and interaction with DNA ligase I (23) results in single-nucleotide BER. Alternatively, as we demonstrate here, a pol beta -dependent excision reaction may switch repair to the long patch BER subpathway. In conclusion, whereas multiple pathways function in BER, pol beta -dependent DNA repair operates in mammalian cells for both single-nucleotide BER and long patch BER.

    ACKNOWLEDGEMENTS

We thank David Winter and Rob Stierum for critical reading of the manuscript. We appreciate the interaction with the Danish Center for Molecular Gerontology.

    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.

§ To whom correspondence should be addressed. Tel.: 410-558-8562; Fax.: 410-558-8157; E-mail: dianovg{at}grc.nia.nih.gov.

2 S. H. Wilson, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: AP sites, apurinic/apyrimidinic sites, abasic sites; BER, base excision repair; PCNA, proliferating cell nuclear antigen; FEN1, flap endonuclease; pol beta  and pol delta , DNA polymerases beta  and delta ; dRP, 5'-deoxyribose phosphate; dRP-oligo, 5'-deoxyribose phosphate oligonucleotide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
  1. Lindahl, T. (1993) Nature 362, 709-715[CrossRef][Medline] [Order article via Infotrieve]
  2. Lindahl, T., Satoh, M. S., and Dianov, G. (1995) Philos. Trans. R. Soc. Lond. B Biol. Sci. 347, 57-62[Medline] [Order article via Infotrieve]
  3. Wilson, S. H. (1998) Mutat. Res. 407, 203-215[Medline] [Order article via Infotrieve]
  4. Matsumoto, Y., Kim, K., and Bogenhagen, D. F. (1994) Mol. Cell. Biol. 14, 6187-6197[Abstract]
  5. Frosina, G., Fortini, P., Rossi, O., Carrozzino, F., Raspaglio, G., Cox, L. S., Lane, D. P., Abbondandolo, A., and Dogliotti, E. (1996) J. Biol. Chem. 271, 9573-9578[Abstract/Free Full Text]
  6. Jonsson, Z. O., and Hubscher, U. (1997) BioEssays 19, 967-975[Medline] [Order article via Infotrieve]
  7. Klungland, A., and Lindahl, T. (1997) EMBO J. 16, 3341-3348[Abstract/Free Full Text]
  8. Li, X., Li, J., Harrington, J., Lieber, M. R., and Burgers, P. M. (1995) J. Biol. Chem. 270, 22109-22112[Abstract/Free Full Text]
  9. Kim, K., Biade, S., and Matsumoto, Y. (1998) J. Biol. Chem. 273, 8842-8848[Abstract/Free Full Text]
  10. Gary, R., Kim, K., Cornelius, H. L., Park, M. S., and Matsumoto, Y. (1999) J. Biol. Chem. 274, 4354-4363[Abstract/Free Full Text]
  11. Biade, S., Sobol, R. W., Wilson, S. H., and Matsumoto, Y. (1998) J. Biol. Chem. 273, 898-902[Abstract/Free Full Text]
  12. Sobol, R. W., Horton, J. K., Kuhn, R., Gu, H., Singhal, R. K., Prasad, R., Rajewsky, K., and Wilson, S. H. (1996) Nature 379, 183-186[CrossRef][Medline] [Order article via Infotrieve]
  13. Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M. L. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3855-3859[Abstract]
  14. Kumar, A., Widen, S., Williams, K., Kedar, P., Karpel, R., and Wilson, S. (1990) J. Biol. Chem. 265, 2124-2131[Abstract/Free Full Text]
  15. Dianov, G., Bischoff, C., Piotrowski, J., and Bohr, V. A. (1998) J. Biol. Chem. 273, 33811-33816[Abstract/Free Full Text]
  16. Singhal, R. K., Prasad, R., and Wilson, S. H. (1995) J. Biol. Chem. 270, 949-957[Abstract/Free Full Text]
  17. Fortini, P., Pascucci, B., Parlanti, E., Sobol, R. W., Wilson, S. H., and Dogliotti, E. (1998) Biochemistry 37, 3575-3580[CrossRef][Medline] [Order article via Infotrieve]
  18. DeMott, M. S., Shen, B., Park, M., Bambara, R. A., and Zingman, S. (1996) J. Biol. Chem. 47, 30068-30076[CrossRef]
  19. Waters, T., Gallinari, P., and Jiricni, J. (1999) J. Biol. Chem. 274, 67-74[Abstract/Free Full Text]
  20. Bennett, R. A. O., Wilson, D. M., 3rd, Wong, D., and Demple, B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7166-7169[Abstract/Free Full Text]
  21. Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702[Medline] [Order article via Infotrieve]
  22. Piersen, C. E., Prasad, R., Wilson, S. H., and Lloyd, R. S. (1996) J. Biol. Chem. 271, 17811-17815[Abstract/Free Full Text]
  23. Prasad, R., Singhal, R. K., Srivastava, D. K., Molina, J. T., Tomkinson, A. E., and Wilson, S. H. (1996) J. Biol. Chem. 271, 16000-16007[Abstract/Free Full Text]


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