Human Thymine DNA Glycosylase Binds to Apurinic Sites in DNA but Is Displaced by Human Apurinic Endonuclease 1 (1999)

In vitro, following the removal of thymine from a G·T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S 6-methylthioguanine (SMeG) or opposite any of the remaining natural DNA bases. It will therefore bind to apurinic sites formed by spontaneous depurination, chemical attack, or other glycosylases. In the absence of magnesium, the rate of dissociation of the glycosylase from such complexes is so slow (k off 1.8 − 3.6 × 10−5 s−1; i.e. half-life between 5 and 10 h) that each molecule of glycosylase removes essentially only one molecule of thymine. In the presence of magnesium, the dissociation rates of the complexes with C·AP andSMeG·AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C·U and SMeG·T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G·AP sites sufficiently to allow it to remove more than one thymine from G·T mismatches. The bound thymine DNA glycosylase prevents human apurinic endonuclease 1 (HAP1) cutting the apurinic site, so unless the glycosylase was displaced, the repair of apurinic sites would be very slow. However, HAP1 significantly increases the rate of dissociation of thymine DNA glycosylase from apurinic sites, presumably through direct interaction with the bound glycosylase. This effect is concentration-dependent and at the probable normal concentration of HAP1 in cells the dissociation would be fast. This interaction couples the first step in base excision repair, the glycosylase, to the second step, the apurinic endonuclease. The other proteins involved in base excision repair, polymerase β, XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site. In vitro, following the removal of thymine from a G·T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S 6-methylthioguanine (SMeG) or opposite any of the remaining natural DNA bases. It will therefore bind to apurinic sites formed by spontaneous depurination, chemical attack, or other glycosylases. In the absence of magnesium, the rate of dissociation of the glycosylase from such complexes is so slow (k off 1.8 − 3.6 × 10−5 s−1; i.e. half-life between 5 and 10 h) that each molecule of glycosylase removes essentially only one molecule of thymine. In the presence of magnesium, the dissociation rates of the complexes with C·AP andSMeG·AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C·U and SMeG·T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G·AP sites sufficiently to allow it to remove more than one thymine from G·T mismatches. The bound thymine DNA glycosylase prevents human apurinic endonuclease 1 (HAP1) cutting the apurinic site, so unless the glycosylase was displaced, the repair of apurinic sites would be very slow. However, HAP1 significantly increases the rate of dissociation of thymine DNA glycosylase from apurinic sites, presumably through direct interaction with the bound glycosylase. This effect is concentration-dependent and at the probable normal concentration of HAP1 in cells the dissociation would be fast. This interaction couples the first step in base excision repair, the glycosylase, to the second step, the apurinic endonuclease. The other proteins involved in base excision repair, polymerase β, XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site. S 6-methylthioguanine apurinic/apyrimidinic site human apurinic endonuclease 1 mismatch-specific uracil DNA glycosylase thymine DNA glycosylase. In mammalian cells, 2–7% of the total cytosine is methylated. Spontaneous deamination of 5-methylcytosine, which is somewhat faster than cytosine (1Ehrlich M. Zhang X.Y. Inamdar N.M. Mutat. Res. 1990; 238: 277-286Crossref PubMed Scopus (100) Google Scholar), generates G·T mispairs in DNA. The repair of these G·T mismatches is initiated by thymine DNA glycosylase which excises the mismatched thymine (2Wiebauer K. Jiricny J. Nature. 1989; 339: 234-236Crossref PubMed Scopus (152) Google Scholar, 3Neddermann P. Jiricny J. J. Biol. Chem. 1993; 268: 21218-21224Abstract Full Text PDF PubMed Google Scholar). 5-Methylcytosine occurs almost exclusively in the sequence MeCpG, and in keeping with its proposed role in the repair of G·T mispairs resulting from the deamination of 5-methylcytosine, thymine DNA glycosylase shows a strong preference for removal of thymine from CpG·T sequences (4Griffin S. Branch P. Xu Y.-Z. Karran P. Biochemistry. 1994; 33: 4787-4793Crossref PubMed Scopus (109) Google Scholar, 5Sibghat-Ullah Day III, R.S. Biochemistry. 1995; 34: 6869-6875Crossref PubMed Scopus (14) Google Scholar, 6Sibghat-Ullah Gallinari P. Xu Y.-Z. Goodman M.F. Bloom L.B. Jiricny J. Day III, R.S. Biochemistry. 1996; 35: 12926-12932Crossref PubMed Scopus (82) Google Scholar, 7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The human enzyme has been cloned and overexpressed (8Neddermann P. Gallinari P. Lettieri T. Schmid D. Truong O. Hsuan J.J. Wiebauer K. Jiricny J. J. Biol. Chem. 1996; 271: 12767-12774Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) and has been shown to belong to a family of uracil DNA glycosylases that remove uracil from G·U base pairs but that are distinct from the general uracil DNA glycosylase enzyme (9Gallinari P. Jiricny J. Nature. 1996; 383: 735-738Crossref PubMed Scopus (183) Google Scholar). Thymine DNA glycosylase removes uracil from G·U base pairs more rapidly than it removes thymine from G·T base pairs (10Neddermann P. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1642-1646Crossref PubMed Scopus (137) Google Scholar) and can also remove uracil from C·U, T·U, and A·U base pairs (7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and may therefore provide a backup function to the general uracil DNA glycosylase. The glycosylase also removes thymine from base pairs with S 6-methylthioguanine (SMeG)1 that are thought to occur in the DNA of cells treated with the drug 6-thioguanine (7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 11Swann P.F. Waters T.R. Moulton D.C. Xu Y.-Z. Zheng Q. Edwards M. Mace R. Science. 1996; 273: 1109-1111Crossref PubMed Scopus (350) Google Scholar). Kinetic studies in our laboratory showed that each molecule of thymine DNA glycosylase can remove only one thymine molecule because the glycosylase remains bound to the apurinic site it produces (7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Since this strong binding may have physiological significance, it has been studied in more detail.Removal of an incorrect base by a DNA glycosylase is the first step of base excision repair (reviewed in Refs. 12Seeberg E. Eide L. Bjørås M. Trends Biochem. Sci. 1995; 20: 391-397Abstract Full Text PDF PubMed Scopus (465) Google Scholar and 13Krokan H.E. Standal R. Slupphaug G. Biochem. J. 1997; 325: 1-16Crossref PubMed Scopus (722) Google Scholar), and one possible role of the bound thymine DNA glycosylase might be to recruit the enzymes needed to complete the repair process. Removal of the base is followed by cleavage of the phosphodiester bond 5′ to the abasic sugar (Fig. 1). In humans, this reaction is most probably carried out by the apurinic endonuclease, HAP1 (also known as APE, APEX, and Ref-1), as this enzyme is responsible for around 95% of all incisions at apurinic sites in HeLa cell extracts (14Kane C.M. Linn S. J. Biol. Chem. 1981; 256: 3405-3414Abstract Full Text PDF PubMed Google Scholar) and can function satisfactorily in reconstituted in vitro base excision repair systems (15Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 16Nicholl I.D. Nealon K. Kenny M.K. Biochemistry. 1997; 36: 7557-7566Crossref PubMed Scopus (77) Google Scholar). Following cleavage of the sugar-phosphate backbone, DNA polymerase β then removes the deoxyribose 5′-phosphate and fills the single base gap (17Wiebauer K. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5842-5845Crossref PubMed Scopus (230) Google Scholar, 18Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 19Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar). Ligation of the nick completes repair and is believed to be carried out by DNA ligase III (15Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar), which is present in cells as a heterodimer with XRCC1 (20Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar). The XRCC1 may act to ensure that polymerase β adds only one nucleotide (15Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). The strong binding of thymine DNA glycosylase to apurinic sites suggested that it might recruit the other proteins involved in base excision repair. Consequently we investigated possible interactions between the thymine DNA glycosylase bound to the apurinic site and the other proteins involved in base excision repair. We found that repair cannot take place while the thymine DNA glycosylase is bound to the apurinic site, and so the extremely slow dissociation of thymine DNA glycosylase from these sites would greatly inhibit repair. However, the apurinic endonuclease HAP1, which catalyzes the second step in base excision repair, can displace the bound thymine DNA glycosylase. This effect, which couples the first and second steps in base excision repair, is concentration-dependent and would be expected to be significant at the concentrations of HAP1 reported in mammalian cells.DISCUSSIONHaving recently found (7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) that each molecule of thymine DNA glycosylase can only remove one thymine because the glycosylase binds so tightly to the G·AP product of the reaction that it is unable to react with another G·T mismatch, we investigated the interaction of thymine DNA glycosylase with apurinic sites in DNA in more detail. We found that the glycosylase can bind to DNA containing an apurinic site opposite guanine, S 6-methylthioguanine (7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), cytosine, thymine, and adenine (Fig. 2). Thus thymine DNA glycosylase shows no absolute preference for the base opposite the apurinic site but seems only to require the apurinic site itself. Furthermore, thymine DNA glycosylase binds to all apurinic sites with a relatively low K d. These findings suggest that thymine DNA glycosylase may bind to apurinic sites in the cell produced by the following: (i) spontaneous depurination; (ii) the action of alkylating agents and other chemicals that react with the bases in DNA to produce adducts with unstable glycosidic bonds, such as 3-alkyladenine or 7-methylguanine (27Lawley P.D. Prog. Nucleic Acids Res. Mol. Biol. 1966; 5: 89-131Crossref PubMed Scopus (280) Google Scholar); and, (iii) the action of other DNA glycosylases such as uracil DNA glycosylase and the glycosylases that remove modified bases (28Singer B. Hang B. Chem. Res. Toxicol. 1997; 10: 713-732Crossref PubMed Scopus (126) Google Scholar).We have measured the dissociation rates for glycosylase-apurinic DNA complexes using a band shift assay that has been used previously for measuring dissociation rates of protein-nucleic acid complexes (23Carey J. Methods Enzymol. 1991; 208: 103-117Crossref PubMed Scopus (318) Google Scholar, 29Werner M. Biochemistry. 1991; 30: 5832-5838Crossref PubMed Scopus (17) Google Scholar,30Long K.S. Crothers D.M. Biochemistry. 1995; 34: 8885-8895Crossref PubMed Scopus (104) Google Scholar). The absolute k off values obtained here need to be confirmed using other techniques and should be treated with caution. However, the dissociation rates are consistent with the results for the reaction of thymine DNA glycosylase (see Fig. 3 and discussion below). In EDTA buffer, the dissociation rates of complexes between thymine DNA glycosylase and all three apurinic containing DNA duplexes tested in Fig. 3 are extremely slow (1.8–3.6 × 10−5 s−1; see Table I) and correspond to half-lives for the complexes of between 5 and 10 h. Assuming a typical second-order association rate (k on) of 107m−1 s−1, these results imply that the binding constants for thymine DNA glycosylase to these apurinic sites in DNA are between 2 and 4 pm. These results confirm our previous finding that the thymine DNA glycosylase action is limited by extremely slow product release (7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The E. coli mismatch-specific uracil glycosylase, MUG, which is a homologue of thymine DNA glycosylase (9Gallinari P. Jiricny J. Nature. 1996; 383: 735-738Crossref PubMed Scopus (183) Google Scholar), also binds to apurinic sites in DNA (31Barrett T.E. Savva R. Panayotou G. Barlow T. Brown T. Jiricny J. Pearl L.H. Cell. 1998; 92: 117-129Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), although the reported K d (6 nm) for the binding of MUG to an apurinic site opposite guanine is a thousand times larger than the value obtained here for human thymine DNA glycosylase.Thymine DNA glycosylase does not require magnesium, and initially we followed the practice of previous authors (2Wiebauer K. Jiricny J. Nature. 1989; 339: 234-236Crossref PubMed Scopus (152) Google Scholar, 4Griffin S. Branch P. Xu Y.-Z. Karran P. Biochemistry. 1994; 33: 4787-4793Crossref PubMed Scopus (109) Google Scholar, 6Sibghat-Ullah Gallinari P. Xu Y.-Z. Goodman M.F. Bloom L.B. Jiricny J. Day III, R.S. Biochemistry. 1996; 35: 12926-12932Crossref PubMed Scopus (82) Google Scholar, 8Neddermann P. Gallinari P. Lettieri T. Schmid D. Truong O. Hsuan J.J. Wiebauer K. Jiricny J. J. Biol. Chem. 1996; 271: 12767-12774Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) and carried out the thymine DNA glycosylase experiments in buffer containing EDTA. As the following step in base excision repair, cutting of the apurinic site by the apurinic endonuclease HAP1 requires at least 0.1 mm magnesium for maximum activity (32Barzilay G. Mol C.D. Robson C.N. Walker L.J. Cunningham R.P. Tainer J.A. Hickson I.D. Nat. Struct. Biol. 1995; 2: 561-568Crossref PubMed Scopus (120) Google Scholar), the dissociation rates were also measured in the presence of 2 mm magnesium. Magnesium had little effect upon the rate of dissociation of thymine DNA glycosylase from G·AP DNA (only a 60% increase ink off), but it increased the rate of dissociation of thymine DNA glycosylase from SMeG·AP and C·AP sites in DNA by 20- and 28-fold, respectively (Table I). This increase in the dissociation rate produced by magnesium has a great influence on the reaction of thymine DNA glycosylase with DNA containing aSMeG·T or a C·U mismatch allowing it to remove more than a stoichiometric amount of thymine from SMeG·T and uracil from C·U mismatches (Fig. 3, F and G). In contrast, magnesium had little effect on the dissociation of thymine DNA glycosylase from a G·AP site in DNA, so even in the presence of magnesium the glycosylase could remove only a stoichiometric amount of the mismatched thymine or uracil from DNA containing a G·T or a G·U mismatch. These results suggest that in the presence of magnesium and with limiting thymine DNA glycosylase, both C·U mismatches andSMeG·T mismatches would be better substrates for the glycosylase than G·T DNA. This is probably irrelevant to the repair of C·U base pairs because they would occur very rarely in cells, and it is almost certain that in vivo uracil would be removed from C·U by the more efficient and abundant uracil DNA glycosylase. However, thymine DNA glycosylase is the only glycosylase known to be able to remove thymine from SMeG·T base pairs so the reasonably rapid attack of thymine DNA glycosylase onSMeG·T mismatches may be important in regard to the cytotoxicity of 6-thioguanine (11Swann P.F. Waters T.R. Moulton D.C. Xu Y.-Z. Zheng Q. Edwards M. Mace R. Science. 1996; 273: 1109-1111Crossref PubMed Scopus (350) Google Scholar).Glycosylases mediate the first step in the base excision repair pathway. The apurinic sites produced are then cut at the apurinic site by an apurinic endonuclease, probably HAP1 (reviewed in Ref. 33Barzilay G. Hickson I.D. BioEssays. 1995; 17: 713-719Crossref PubMed Scopus (196) Google Scholar). We had expected that the bound thymine DNA glycosylase might recruit HAP1 and thus facilitate the cleavage of the apurinic site, but unexpectedly it was found that neither HAP1 nor Endonuclease IV can cut at the apurinic site while thymine DNA glycosylase is bound to it (Fig. 5,A and B). Thus dissociation of the thymine DNA glycosylase from the apurinic site is a prerequisite for complete repair to take place (Fig. 7). HAP1 increases the rate of this dissociation. This increases the removal of thymine from G·T mismatches, because the displacement allows turnover of the glycosylase (Fig. 5) and accelerates the repair because it allows HAP1 to cut the apurinic site, which in turn allows the other enzymes involved in base excision repair to refill the gap in the DNA. The displacement of thymine DNA glycosylase from the apurinic site appears to be specific to HAP1 since Endonuclease IV, a type II endonuclease from E. coli, has no effect on the glycosylase reaction (Fig.5 A). No similar interaction between a DNA glycosylase and HAP1 has been reported before. The increase induced by 6 nmHAP1 in the rate of turnover of 6 nm thymine DNA glycosylase was quite small, but the increase is concentration-dependent and at 600 nm HAP1 a much larger increase in turnover was induced. From the purification of HAP1 from HeLa cells (14Kane C.M. Linn S. J. Biol. Chem. 1981; 256: 3405-3414Abstract Full Text PDF PubMed Google Scholar), we estimate that the concentration of HAP1 is about 0.1–1 mm (a similar figure is given in Ref. 34Bennett R.A.O. Wilson III, D.M. Wong D. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7166-7169Crossref PubMed Scopus (325) Google Scholar), and at this concentration HAP1 should produce a very substantial increase in the rate of dissociation of thymine DNA glycosylase from apurinic sites. The rate of removal of thymine from G·T mismatches by thymine DNA glycosylase in the presence of such a large concentration of HAP1 should be more than adequate to cope with the rate of deamination of 5-methylcytosine in cells (35Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4273) Google Scholar). The high concentration of HAP1 in cells may, in part, reflect its role in maintaining the redox state of some transcription factors (33Barzilay G. Hickson I.D. BioEssays. 1995; 17: 713-719Crossref PubMed Scopus (196) Google Scholar), but it may also reflect the necessity for a large concentration of HAP1 to dissociate the thymine DNA glycosylase, and conceivably other DNA glycosylases, from apurinic sites.A multiprotein complex that fully repairs uracil sites in DNA has been purified suggesting that components of base excision repair may exist as a "repairosome" that can carry out complete repair in a concerted manner (36Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Kubota et al. (15Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar) reconstitutedin vitro the repair of uracil in DNA using the human proteins uracil DNA glycosylase, HAP1, polymerase β, XRCC1, and DNA ligase III. In an analogous experiment, we reconstituted in vitro repair of DNA containing a G·T mismatch using thymine DNA glycosylase, HAP1, polymerase β, XRCC1, and DNA ligase III. Our present data do not support the view that these proteins act as a repair complex analogous to that seen in nucleotide excision repair (reviewed in Ref. 37Wood R.D. J. Biol. Chem. 1997; 272: 23465-23468Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Although this combination of proteins could completely repair the G·T mismatch, the rate of the glycosylase reaction was no greater than that seen with thymine DNA glycosylase and HAP1 alone (Fig. 7) showing that these other repair proteins are not able to cooperate with HAP1 in the displacement of the glycosylase from the apurinic site. Furthermore, there was no evidence from band shift assays that HAP1, which is the only one of these proteins that had any effect on the dissociation of the thymine DNA glycosylase from the apurinic site, formed a complex with the thymine DNA glycosylase bound to the apurinic site (Fig. 6).The interaction between thymine DNA glycosylase and HAP1 would loosely coordinate the first and second step of base excision repair and appears similar to the recently reported interaction between HAP1 and DNA polymerase β R.A.O. Wilson III, D.M. Wong D. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7166-7169Crossref PubMed Scopus (325) Google Scholar) that would loosely coordinate the second and interaction between polymerase β and a complex of DNA ligase III and XRCC1 has also been (15Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). The effect of these interactions might be to all the steps in the pathway. However, this does not the glycosylase binds so to apurinic sites. the site were not by the bound glycosylase it would be very rapidly cut by HAP1, which is a relatively enzyme Wilson S.H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), and so the binding of the glycosylase to apurinic sites than repair. from the that in human the amount of HAP1 measured in some cells is quite and in some cells HAP1 is and thus may not be for DNA repair Wilson Res. 1995; Google Scholar). could that in cells there was a of HAP1, the binding of thymine DNA glycosylase to apurinic sites might to the apurinic site or might act as a to DNA or transcription and opposite the apurinic site. it might act as a to the cell DNA repair can be in might act as the for In mammalian cells, 2–7% of the total cytosine is methylated. Spontaneous deamination of 5-methylcytosine, which is somewhat faster than cytosine (1Ehrlich M. Zhang X.Y. Inamdar N.M. Mutat. Res. 1990; 238: 277-286Crossref PubMed Scopus (100) Google Scholar), generates G·T mispairs in DNA. The repair of these G·T mismatches is initiated by thymine DNA glycosylase which excises the mismatched thymine (2Wiebauer K. Jiricny J. Nature. 1989; 339: 234-236Crossref PubMed Scopus (152) Google Scholar, 3Neddermann P. Jiricny J. J. Biol. Chem. 1993; 268: 21218-21224Abstract Full Text PDF PubMed Google Scholar). 5-Methylcytosine occurs almost exclusively in the sequence MeCpG, and in keeping with its proposed role in the repair of G·T mispairs resulting from the deamination of 5-methylcytosine, thymine DNA glycosylase shows a strong preference for removal of thymine from CpG·T sequences (4Griffin S. Branch P. Xu Y.-Z. Karran P. Biochemistry. 1994; 33: 4787-4793Crossref PubMed Scopus (109) Google Scholar, 5Sibghat-Ullah Day III, R.S. Biochemistry. 1995; 34: 6869-6875Crossref PubMed Scopus (14) Google Scholar, 6Sibghat-Ullah Gallinari P. Xu Y.-Z. Goodman M.F. Bloom L.B. Jiricny J. Day III, R.S. Biochemistry. 1996; 35: 12926-12932Crossref PubMed Scopus (82) Google Scholar, 7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The human enzyme has been cloned and overexpressed (8Neddermann P. Gallinari P. Lettieri T. Schmid D. Truong O. Hsuan J.J. Wiebauer K. Jiricny J. J. Biol. Chem. 1996; 271: 12767-12774Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) and has been shown to belong to a family of uracil DNA glycosylases that remove uracil from G·U base pairs but that are distinct from the general uracil DNA glycosylase enzyme (9Gallinari P. Jiricny J. Nature. 1996; 383: 735-738Crossref PubMed Scopus (183) Google Scholar). Thymine DNA glycosylase removes uracil from G·U base pairs more rapidly than it removes thymine from G·T base pairs (10Neddermann P. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1642-1646Crossref PubMed Scopus (137) Google Scholar) and can also remove uracil from C·U, T·U, and A·U base pairs (7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and may therefore provide a backup function to the general uracil DNA glycosylase. The glycosylase also removes thymine from base pairs with S 6-methylthioguanine (SMeG)1 that are thought to occur in the DNA of cells treated with the drug 6-thioguanine (7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 11Swann P.F. Waters T.R. Moulton D.C. Xu Y.-Z. Zheng Q. Edwards M. Mace R. Science. 1996; 273: 1109-1111Crossref PubMed Scopus (350) Google Scholar). Kinetic studies in our laboratory showed that each molecule of thymine DNA glycosylase can remove only one thymine molecule because the glycosylase remains bound to the apurinic site it produces (7Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Since this strong binding may have physiological significance, it has been studied in more detail. Removal of an incorrect base by a DNA glycosylase is the first step of base excision repair (reviewed in Refs. 12Seeberg E. Eide L. Bjørås M. Trends Biochem. Sci. 1995; 20: 391-397Abstract Full Text PDF PubMed Scopus (465) Google Scholar and 13Krokan H.E. Standal R. Slupphaug G. Biochem. J. 1997; 325: 1-16Crossref PubMed Scopus (722) Google Scholar), and one possible role of the bound thymine DNA glycosylase might be to recruit the enzymes needed to complete the repair process. Removal of the base is followed by cleavage of the phosphodiester bond 5′ to the abasic sugar (Fig. 1). In humans, this reaction is most probably carried out by the apurinic endonuclease, HAP1 (also known as APE, APEX, and Ref-1), as this enzyme is responsible for around 95% of all incisions at apurinic sites in HeLa cell extracts (14Kane C.M. Linn S. J. Biol. Chem. 1981; 256: 3405-3414Abstract Full Text PDF PubMed Google Scholar) and can function satisfactorily in reconstituted in vitro base excision repair systems (15Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 16Nicholl I.D. Nealon K. Kenny M.K. Biochemistry. 1997; 36: 7557-7566Crossref PubMed Scopus (77) Google Scholar). Following cleavage of the sugar-phosphate backbone, DNA polymerase β then removes the deoxyribose 5′-phosphate and fills the single base gap (17Wiebauer K. Jiricny J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5842-5845Crossref PubMed Scopus (230) Google Scholar, 18Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 19Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar). Ligation of the nick completes repair and is believed to be carried out by DNA ligase III (15Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar), which is present in cells as a heterodimer with XRCC1 (20Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar). The XRCC1 may act to ensure that polymerase β adds only one nucleotide (15Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). The strong binding of thymine DNA glycosylase to apurinic sites suggested that it might recruit the other proteins involved in base excision repair. Consequently we investigated possible interactions between the thymine DNA glycosylase bound to the apurinic site and the other proteins involved in base excision repair. We found that repair cannot take place