FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2051444751> ?p ?o ?g. }

• W2051444751 endingPage "7893" @default.
• W2051444751 startingPage "7888" @default.
• W2051444751 abstract "Apurinic/apyrimidinic (AP) sites occur frequently in DNA as a result of spontaneous base loss or following removal of a damaged base by a DNA glycosylase. The action of many AP endonuclease enzymes at abasic sites in DNA leaves a 5′-deoxyribose phosphate (dRP) residue that must be removed during the base excision repair process. This 5′-dRP group may be removed by AP lyase enzymes that employ a β-elimination mechanism. This β-elimination reaction typically involves a transient Schiff base intermediate that can react with sodium borohydride to trap the DNA-enzyme complex. With the use of this assay as well as direct 5′-dRP group release assays, we show that T4 DNA ligase, a representative ATP-dependent DNA ligase, contains AP lyase activity. The AP lyase activity of T4 DNA ligase is inhibited in the presence of ATP, suggesting that the adenylated lysine residue is part of the active site for both the ligase and lyase activities. A model is proposed whereby the AP lyase activity of DNA ligase may contribute to the repair of abasic sites in DNA. Apurinic/apyrimidinic (AP) sites occur frequently in DNA as a result of spontaneous base loss or following removal of a damaged base by a DNA glycosylase. The action of many AP endonuclease enzymes at abasic sites in DNA leaves a 5′-deoxyribose phosphate (dRP) residue that must be removed during the base excision repair process. This 5′-dRP group may be removed by AP lyase enzymes that employ a β-elimination mechanism. This β-elimination reaction typically involves a transient Schiff base intermediate that can react with sodium borohydride to trap the DNA-enzyme complex. With the use of this assay as well as direct 5′-dRP group release assays, we show that T4 DNA ligase, a representative ATP-dependent DNA ligase, contains AP lyase activity. The AP lyase activity of T4 DNA ligase is inhibited in the presence of ATP, suggesting that the adenylated lysine residue is part of the active site for both the ligase and lyase activities. A model is proposed whereby the AP lyase activity of DNA ligase may contribute to the repair of abasic sites in DNA. DNA repair pathways have evolved to process a wide range of chemically distinct lesions in DNA (1Friedberg E.C. Walker G.C. Seide W. DNA Repair and Mutagenesis. ASM Press, Washington, D.C.1995Google Scholar). One of the most common types of damage is spontaneous or enzymatic hydrolysis of the N-glycosidic bond between a DNA base and the sugar phosphate backbone generating an abasic site (AP site). 1The abbreviations used are: AP, apurinic/apyrimidinic; UDG, uracil DNA glycosylase; dRP, 2′-deoxyribose 5′-phosphate; FPG, formamidopyrimidine glycosylase; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis. AP sites are highly mutagenic and require rapid and efficient repair. AP sites are processed by a base excision repair pathway that is frequently initiated by the action of a class II AP endonuclease that cleaves the DNA backbone adjacent to the lesion to produce 3′-OH and 2′-deoxyribose 5′-phosphate termini (2Barzilay G. Hickson I.D. BioEssays. 1995; 17: 713-719Crossref PubMed Scopus (196) Google Scholar). The latter residue, referred to as a 5′-dRP moiety, is relatively alkali labile and can be removed by AP lyases that facilitate β-elimination. Most enzymes demonstrated to have dRPase activity operate through this lyase mechanism, although some, such as the Escherichia coli recJ protein, catalyze hydrolysis (3Dianov G. Sedgwick B. Daly G. Olsson M. Lovett S. Lindahl T. Nucleic Acids Res. 1994; 22: 993-998Crossref PubMed Scopus (88) Google Scholar). The two classes of dRPase enzymes can be distinguished by the fact that the lyase mechanism frequently involves formation of a transient Schiff base intermediate in which an amino group on the enzyme is covalently bound to the DNA. This lyase mechanism, first proposed for E. coli endonuclease III (4Kow Y.W. Wallace S.S. Biochemistry. 1987; 26: 8200-8206Crossref PubMed Scopus (155) Google Scholar), provides a simple method to identify a polypeptide with AP lyase activity because the Schiff base intermediate can be trapped in a stable form by reaction with a strong reducing agent, such as NaBH4 or NaBH3CN. Thus, with an appropriate radioactively labeled substrate, the label can be transferred to the AP lyase enzyme. This borohydride trapping has been documented for several repair enzymes (5Graves R.J. Felzenszwalb I. Laval J. O'Connor T.R. J. Biol. Chem. 1992; 267: 14429-14435Abstract Full Text PDF PubMed Google Scholar, 6Dodson M.L. Schrock R.D.I. Lloyd R.S. Biochemistry. 1993; 32: 8284-8290Crossref PubMed Scopus (136) Google Scholar, 7Tchou J. Grollman A.P. J. Biol. Chem. 1995; 270: 11671-11677Crossref PubMed Scopus (127) Google Scholar, 8Sun B. Latham K.A. Dodson M.L. Lloyd R.S. J. Biol. Chem. 1995; 270: 19501-19508Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 9Nash H.M. Bruner S.D. Scharer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar) and, more recently, for DNA pol β (10Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (649) Google Scholar, 11Piersen C.E. Prasad R. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1996; 271: 17811-17815Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The combined action of AP endonuclease and AP lyase leaves a one-nucleotide gap that is filled by a DNA polymerase. The final step in repair involves DNA strand sealing by DNA ligase. The mechanism of DNA ligase involves covalent modification of the enzyme by adenylation, transfer of the AMP residue in a phosphoanhydride linkage to the 5′-phosphate of nicked DNA, followed by resealing of the DNA strand driven by the energy of AMP hydrolysis. We recently characterized a mtDNA ligase as part of an effort to reconstitute repair of AP sites using mitochondrial enzymes (12Pinz K.G. Bogenhagen D.F. Mol. Cell. Biol. 1998; 18 (in press)Crossref PubMed Scopus (164) Google Scholar). The size, template specificity, and immunological properties of the mtDNA ligase suggested that this was a form of DNA ligase III. In the course of this work we found that mtDNA ligase is active on a DNA substrate containing an AP site incised on the 5′ side by a class II AP endonuclease. Our observations prompted a detailed investigation of the action of T4 DNA ligase as a prototype for ATP-dependent DNA ligases at AP sites in DNA. In this paper we show that in the presence of ATP, T4 DNA ligase is able to reseal an incised AP site. In the absence of ATP, T4 DNA ligase acts as an AP lyase to facilitate a β-elimination reaction that leads to removal of the 5′-dRP residue. A model is presented whereby an intrinsic AP lyase activity in DNA ligase may facilitate repair of AP sites. mtDNA ligase, mitochondrial AP endonuclease, and DNA ligase I were purified from Xenopus ovary tissue as described (12Pinz K.G. Bogenhagen D.F. Mol. Cell. Biol. 1998; 18 (in press)Crossref PubMed Scopus (164) Google Scholar). T4 DNA ligase was obtained from Boehringer Mannheim. T7 DNA ligase was a gift from Dr. J. Dunn (Brookhaven National Laboratory). Variable quantities of both preparations of bacteriophage DNA ligase were subjected to SDS-PAGE analysis (13Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar) in parallel with standard proteins of known concentration. The gels were stained with Coomassie Blue to confirm that both preparations were essentially homogeneous and to permit estimation of protein concentrations using densitometry of the stained gels. Radiochemicals were purchased from ICN Radiochemicals. Uracil DNA glycosylase (UDG) was obtained from Epicentre Technologies (HK-UNG). FPG protein was a gift from J. Tchou and A. P. Grollman (SUNY-Stony Brook). Sodium borohydride and sodium thioglycolate were obtained from Sigma-Aldrich. Other reagent grade chemicals were obtained from Sigma-Aldrich or Fisher. The Poros Q 4.6 × 50-mm column used for anion exchange HPLC was obtained from Perceptive Biosystems. Oligonucleotides were either synthesized by the phosphoramidite method at the SUNY-Stony Brook Oligonucleotide Synthesis Facility or were obtained from Operon. A continuous duplex oligonucleotide was prepared by annealing a 5′-32P kinase-labeled 32-mer (5′-CATGGGCCGACATGAUCAAGCTTGAGGCCAAG) to a complementary oligonucleotide (5′-TCTTGGCCTCAAGCTTGATCATGTCGGCCCATG). Two nicked duplex oligonucleotides were prepared by annealing a 5′-32P kinase-labeled 17-mer (5′-UCAAGCTTGAGGCCAAG; referred to as U17) and either a nonradioactive 15-mer (5′-CATGGGCCGACATGA) or 12-mer (5′-GGGCCGACATGA) to the same complementary strand described above. The 12-mer was used in the experiment in Fig. 7 C to permit better resolution of the reaction product from the initial labeled substrate. Oligonucleotides were phosphorylated using standard procedures (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) and annealed by heating to 80 °C in 0.1m NaCl, 10 mm Tris, pH 8, 1 mm EDTA and slow cooling to 4 °C. Oligonucleotides were treated immediately before use with UDG in 20 mm Hepes, pH 7.5, and diluted into an assay mixture with DNA ligase in 10 mm Hepes, pH 7.5, 1 mm MgCl2, unless otherwise indicated. Borohydride trapping was performed by a modification of published procedures (9Nash H.M. Bruner S.D. Scharer O.D. Kawate T. Addona T.A. Spooner E. Lane W.S. Verdine G.L. Curr. Biol. 1996; 6: 968-980Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 11Piersen C.E. Prasad R. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1996; 271: 17811-17815Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) by including 20 or 50 mmNaBH4 in the binding reaction. After 30 min at 25 °C, the solution was adjusted to contain 6 mm CaCl2and 10 μg/ml micrococcal nuclease. After 20 min of incubation at 37 °C, proteins were precipitated with trichloroacetic acid, analyzed by SDS-polyacrylamide gel electrophoresis (13Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar), and detected by autoradiography or PhosphorImager analysis (Molecular Dynamics). dRPase activity was measured as described (3Dianov G. Sedgwick B. Daly G. Olsson M. Lovett S. Lindahl T. Nucleic Acids Res. 1994; 22: 993-998Crossref PubMed Scopus (88) Google Scholar), and HPLC analysis of products generated in the presence of sodium thioglycolate was performed as described (5Graves R.J. Felzenszwalb I. Laval J. O'Connor T.R. J. Biol. Chem. 1992; 267: 14429-14435Abstract Full Text PDF PubMed Google Scholar, 15Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (261) Google Scholar), except that a Poros Q anion exchange column was used. Our laboratory has studied the base excision repair pathway with nuclear and mitochondrial protein fractions using templates containing precisely positioned single AP sites embedded in covalently closed circular DNA (12Pinz K.G. Bogenhagen D.F. Mol. Cell. Biol. 1998; 18 (in press)Crossref PubMed Scopus (164) Google Scholar, 15Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (261) Google Scholar). These sites are readily cleaved on the 5′ side by class II AP endonuclease to yield a 3′-OH terminus and a 5′-deoxyribose phosphate (dRP) residue. When templates bearing incised AP sites are incubated with DNA ligase in the presence of ATP, the DNA ligases are able to reseal the nicked strand (Fig.1). This reaction has been reported for T4 DNA ligase (16Goffin C. Bailly V. Verly W.G. Nucleic Acids Res. 1987; 15: 8755-8771Crossref PubMed Scopus (47) Google Scholar) but not for eukaryotic DNA ligases. Because ligation of a 5′-dRP moiety directly reverses the action of AP endonuclease, it is counterproductive for repair. It is also a potential confounding factor in efforts to reconstitute repair reactions in vitro, because religation of an abasic site might not be differentiated from actual repair. In the experiment in Fig. 1, we used a substrate with a synthetic analogue of an abasic site, a tetrahydrofuran analogue that has been used extensively in repair studies (15Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (261) Google Scholar, 17Takeshita M. Chang C.-N. Johnson F. Will S. Grollman A.P. J. Biol. Chem. 1987; 262: 10171-10179Abstract Full Text PDF PubMed Google Scholar). This is an analogue of a reduced deoxyribose moiety and is not subject to β-elimination. Experiments presented below show that T4 DNA ligase can also reseal an authentic (nonreduced) AP site. We previously showedXenopus laevis mtDNA ligase can be labeled using a borohydride trapping procedure that is specific for AP lyase activities (12Pinz K.G. Bogenhagen D.F. Mol. Cell. Biol. 1998; 18 (in press)Crossref PubMed Scopus (164) Google Scholar). To determine whether this is a general property of ATP-dependent DNA ligases, we tested T4 and T7 DNA ligases for the ability to react with AP sites in a borohydride trapping assay. This assay employed an oligonucleotide substrate designed to contain a specific U residue adjacent to a nick, referred to as 15-*U17:33 to denote a 15-mer annealed adjacent to a kinase-labeled (*) 17-mer to a 33-mer complementary strand. Treatment of the duplex oligonucleotide with UDG results in a 5′-phosphoryl abasic site. This is an exact model of the substrate that would be generated by action of a class II AP endonuclease. Fig. 2 shows that both T4 DNA ligase and T7 DNA ligase react in this assay. Controls in lanes 3 and 4 of Fig. 2 demonstrate that cross-linking was dependent on the presence of NaBH4 and on the AP site, because the reaction was not observed with a control oligonucleotide containing thymine in place of uracil. We performed a variety of control experiments to characterize the putative AP lyase activity in T4 DNA ligase. The extent of cross-linking with a nicked substrate varies with solution conditions. Cross-linking is reduced at 5 mm MgCl2 in comparison with the standard reaction conducted at 1 mmMgCl2 and is inhibited more than 90% by 1 mmATP (Fig. 3). The standard reaction includes post-treatment with micrococcal nuclease to degrade the oligonucleotide cross-linked to protein by NaBH4. When this nuclease treatment was omitted, the electrophoretic mobility of the major cross-linked protein species was reduced, as expected for a DNA-protein complex (Fig. 3 B). In the absence of micrococcal nuclease treatment a minor cross-linked labeled species with essentially the same gel mobility as unmodified T4 DNA ligase was also observed. This faster migrating species is expected to be formed as an intermediate in the action of an AP lyase, as shown in Fig.4. The initial product of an attack by AP lyase on an AP site is a Schiff base intermediate in which the enzyme is joined to DNA. The AP lyase reaction proceeds with elimination of the DNA from the C3′ position of deoxyribose, producing an enzyme-dRP intermediate with a Schiff base linkage. Both the enzyme-DNA and enzyme-dRP species can be reduced by NaBH4 to generate the doublet of cross-linked species in Fig. 3 B. These borohydride cross-linking results are consistent with the hypothesis that T4 DNA ligase contains AP lyase activity.Figure 4The chemistry of AP lyase action accounts for DNA-enzyme and dRP-enzyme complexes following NaBH4treatment. This scheme is based on those presented for other AP lyase enzymes (7Tchou J. Grollman A.P. J. Biol. Chem. 1995; 270: 11671-11677Crossref PubMed Scopus (127) Google Scholar, 19Dodson M.L. Michaels M.L. Lloyd R.S. J. Biol. Chem. 1994; 269: 32709-32712Abstract Full Text PDF PubMed Google Scholar). The Shiff base reaction scheme requires attack by a free amino group of the AP lyase on the C1′ residue of the deoxyribose. This reactive nitrogen is referred to as N-enz. Other functional groups within the enzyme may assist in the β-elimination reaction as indicated. Covalent intermediates in the Schiff base reaction scheme labeled 1 and 2 may be reduced by borohydride to yield stable species with the protein cross-linked to an oligonucleotide or to a dRP moiety, respectively.View Large Image Figure ViewerDownload (PPT) In other experiments, we found that it was not necessary to present the AP site in the context of a nick in DNA, although this is the preferred substrate. Borohydride trapping was observed when the 15-mer oligonucleotide was omitted from the standard nicked substrate, leaving a 5′-AP site adjacent to single-stranded DNA. We also observed cross-linking to a free oligonucleotide with a 5′-dRP site generated by the action of UDG on the 5′U-17-mer oligonucleotide (5′-32P-UCAAGCTTGAGGCCAAG). The efficiency of borohydride trapping with a single-stranded 5′-AP oligonucleotide was about 50% of that observed with the nicked oligonucleotide substrate. The single-stranded oligonucleotide substrate was used in some experiments described below to simplify preparation of large quantities of substrate for AP lyase assays. The borohydride trapping reaction is a very sensitive probe of AP lyase activity but is not always efficient because it acts on a transient intermediate in the overall AP lyase reaction. For DNA glycosylases that remove a base and attack the AP site in a sequential dual mechanism, such as FPG protein, this borohydride trapping procedure is relatively easy to control. It is more difficult to trap a large fraction of a protein that does not contain an intrinsic glycosylase activity because the NaBH4 required to cross-link the enzyme-DNA Schiff base intermediate can also react with the ring open form of the sugar residue to inactivate the substrate. As an independent assay for AP lyase activity, we monitored the release of free 5′-dRP from DNA as an acid or ethanol-soluble species, as shown in Fig. 5. When reactions were performed with a high concentration of the single stranded 5′-dRP-17-mer oligonucleotide, we found that dRP release was linear with time, showed a clear temperature optimum, and was inhibited by ATP as observed for the borohydride trapping assay. The ethanol-soluble species released by T4 DNA ligase in the presence of thioglycolic acid was observed to have the same chromatographic properties as the product released by FPG protein, which has a well characterized AP lyase activity (Fig.6 and Refs. 5Graves R.J. Felzenszwalb I. Laval J. O'Connor T.R. J. Biol. Chem. 1992; 267: 14429-14435Abstract Full Text PDF PubMed Google Scholar, 7Tchou J. Grollman A.P. J. Biol. Chem. 1995; 270: 11671-11677Crossref PubMed Scopus (127) Google Scholar, and 18Bricteux-Gregoire S. Verly W.G. Nucleic Acids Res. 1989; 17: 6269-6282Crossref PubMed Scopus (9) Google Scholar).Figure 6The putative 5′-dRP product released by DNA ligase has the same chromatographic properties as that released by FPG protein, a well characterized AP lyase. The 5′-32P U17 oligo pretreated with UDG was incubated with either FPG protein as a positive control (A) or with T4 DNA ligase (B) in the presence of 50 mm sodium thioglycolate. The product released by the lyase activity of FPG protein has been shown to react with thioglycolate to generate an anionic species (5Graves R.J. Felzenszwalb I. Laval J. O'Connor T.R. J. Biol. Chem. 1992; 267: 14429-14435Abstract Full Text PDF PubMed Google Scholar, 18Bricteux-Gregoire S. Verly W.G. Nucleic Acids Res. 1989; 17: 6269-6282Crossref PubMed Scopus (9) Google Scholar). Reaction of the β-elimination product with thioglycolate leads to reduction of the aldehyde, blocking the subsequent δ-elimination reaction for FPG protein. The ethanol-soluble reaction products were analyzed by chromatography on a Poros Q HPLC column using the indicated NaCl gradient. The intact oligonucleotide elutes from this column with the 1m NaCl step.View Large Image Figure ViewerDownload (PPT) The borohydride trapping and dRP release assays show that T4 DNA ligase is an authentic AP lyase by the same criteria used to show that DNA pol β possesses AP lyase activity (10Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (649) Google Scholar, 11Piersen C.E. Prasad R. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1996; 271: 17811-17815Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Although T4 DNA ligase is capable of processing multiple AP site substrates (Fig. 5 A), the turnover number is less than 10% of the vigorous rate observed for FPG protein (5Graves R.J. Felzenszwalb I. Laval J. O'Connor T.R. J. Biol. Chem. 1992; 267: 14429-14435Abstract Full Text PDF PubMed Google Scholar). It should be noted that the glycosylase activity of FPG protein is reported to be significantly slower than the AP lyase activity (5Graves R.J. Felzenszwalb I. Laval J. O'Connor T.R. J. Biol. Chem. 1992; 267: 14429-14435Abstract Full Text PDF PubMed Google Scholar). A lower turnover number for the AP lyase activity in T4 DNA ligase may be expected because the enzyme is likely to bind persistently to the gapped substrate generated by AP lyase action on a site previously cleaved by AP endonuclease. We have not yet identified the active site for the DNA ligase-associated AP lyase. The borohydride trapping reaction requires attack on the C1′ residue of deoxyribose by an N-terminal amino group or by an internal lysine (19Dodson M.L. Michaels M.L. Lloyd R.S. J. Biol. Chem. 1994; 269: 32709-32712Abstract Full Text PDF PubMed Google Scholar). The fact that the AP lyase activity is suppressed by ATP suggests that the active site lysine residue that is adenylated in DNA ligase (20Tomkinson A.E. Totty N.F. Ginsburg M. Lindahl T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 400-404Crossref PubMed Scopus (106) Google Scholar) may be involved directly in the nucleophilic attack that promotes β-elimination. This residue is normally in close proximity to the nick in a DNA substrate in the course of a DNA ligation reaction. However, we have not ruled out the possibility that another lysine residue may be involved in this attack, because the deadenylated enzyme may have an altered conformation that interacts differently with DNA substrates containing 5′-dRP residues. It will be particularly interesting to map the active site residue in T7 DNA ligase that reacts in the borohydride trapping reaction because the structure of this enzyme has been determined (21Subramanya H.S. Doherty A.J. Ashford S.R. Wigley D.B. Cell. 1996; 85: 607-615Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). This enzyme has the added advantage that it is relatively small, with only 359 amino acid residues. We have observed AP lyase activity using the borohydride trapping assay for the following four different ATP-dependent DNA ligases: T4 and T7 DNA ligase (Fig. 2), mtDNA ligase (12Pinz K.G. Bogenhagen D.F. Mol. Cell. Biol. 1998; 18 (in press)Crossref PubMed Scopus (164) Google Scholar), and DNA ligase I (data not shown). Because ATP-dependent DNA ligases as a class share structural and functional features (22Lindahl T. Barnes D.E. Annu. Rev. Biochem. 1992; 61: 251-281Crossref PubMed Scopus (187) Google Scholar), it is likely that the presence of AP lyase activity will be conserved in this family. To date we have not been able to document AP lyase activity in bacterial DNA ligases from eitherE. coli or T. aquaticus either in the presence or the absence of their cofactor, NAD (data not shown). The critical question raised by our observations is whether the AP lyase activity associated with T4 DNA ligase plays a significant physiological role. A model for the action of T4 DNA ligase at AP sites is shown in Fig. 7. In living cells, AP sites are very rapidly incised by AP endonuclease to generate the sort of nicked AP substrate we have used in our reactions. The experiments reported here show that T4 DNA ligase can act as an AP lyase at these sites in the absence of ATP. Under these conditions, the deadenylated enzyme cannot seal the nick and instead facilitates β-elimination, leading to loss of the 5′-dRP residue. This produces the single nucleotide gap structure diagramed as species 5 in Fig. 7. This single base gap may be repaired by the action of DNA polymerase and the conventional strand sealing action of DNA ligase. It is also important to consider the action of T4 DNA ligase at incised AP sites in the presence of ATP, because a large fraction of DNA ligase may exist in the adenylated state in vivo. Our results suggest that the adenylated T4 DNA ligase has a reduced ability to promote β-elimination. Instead, when a T4 DNA ligase molecule that is activated by adenylation binds this nicked substrate, it seals the nick to regenerate an internal AP site, as shown in Fig. 1 and diagramed as species 3 in Fig. 7 A. The second product of the ligation reaction is a “disarmed” DNA ligase molecule that is no longer adenylated but is still in contact with the AP site. To test whether T4 DNA ligase is able to incise DNA on the 3′ side of an internal AP site (i.e., without prior action of an AP endonuclease), we performed the experiment in Fig. 7 B. This experiment shows that T4 DNA ligase is clearly capable of strand incision to yield a product with a slightly slower gel mobility than that produced by the well characterized AP lyase of FPG protein. This suggests that T4 DNA ligase is able to promote β-elimination but unlike FPG protein does not efficiently promote δ-elimination. This sort of incision reaction was not observed in Fig. 1 because that experiment employed a reduced AP site analogue. These results suggest that when T4 DNA ligase seals a nick generated by class II AP endonuclease, it may recognize the product as a mistake and employ its lyase activity to reopen the DNA. To test this prediction, we performed the experiment shown in Fig. 7 C. In this experiment, T4 DNA ligase was incubated with a 17-mer oligonucleotide containing a 5′-32P-dRP residue adjacent to a nonradioactive 12-mer. DNA ligase was able to ligate the 12-mer to the 5′-dRP-17 mer to generate a 29-mer with an internal 32P-dRP residue (lane 3of Fig. 7 C). A limited extent of ligation was observed without the addition of exogenous ATP (lane 2), presumably because a fraction of the T4 DNA ligase is purified in an adenylated form. The more efficient ligation in the presence of ATP was followed by incision on the 3′ side of the AP site to produce a labeled 12-mer with a 3′-dRP residue. Thus, the label transfer experiment in Fig.7 C confirms the model for the action of T4 DNA ligase at an AP site in the presence of ATP. The ring open 3′-dRP residue produced by AP lyase cannot be rejoined by DNA ligase due to the 2′-3′-double bond, but the 3′-dRP group would be susceptible to release by class II AP endonuclease. Taken together, these experiments suggest that the role of T4 DNA ligase in base excision repair may not be limited to the final step of strand closure. We thank J. Dunn, F. Johnson, Y. Matsumoto, and B. Weiss for helpful discussion and for comments on the manuscript and J. Tchou and J. Dunn for gifts of E. coli FPG protein and T7 DNA ligase, respectively." @default.
• W2051444751 created "2016-06-24" @default.
• W2051444751 creator A5064165292 @default.
• W2051444751 creator A5064451780 @default.
• W2051444751 date "1998-04-01" @default.
• W2051444751 modified "2023-09-28" @default.
• W2051444751 title "The Action of DNA Ligase at Abasic Sites in DNA" @default.
• W2051444751 cites W1494022099 @default.
• W2051444751 cites W1528920733 @default.
• W2051444751 cites W1561613766 @default.
• W2051444751 cites W1602417853 @default.
• W2051444751 cites W1965158726 @default.
• W2051444751 cites W1992514146 @default.
• W2051444751 cites W1998426722 @default.
• W2051444751 cites W2000605935 @default.
• W2051444751 cites W2007830417 @default.
• W2051444751 cites W2011068146 @default.
• W2051444751 cites W2025823631 @default.
• W2051444751 cites W2046561854 @default.
• W2051444751 cites W2091404614 @default.
• W2051444751 cites W2097950713 @default.
• W2051444751 cites W2100837269 @default.
• W2051444751 cites W2109323414 @default.
• W2051444751 cites W2125735909 @default.
• W2051444751 cites W2174944132 @default.
• W2051444751 cites W2184161959 @default.
• W2051444751 doi "https://doi.org/10.1074/jbc.273.14.7888" @default.
• W2051444751 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9525883" @default.
• W2051444751 hasPublicationYear "1998" @default.
• W2051444751 type Work @default.
• W2051444751 sameAs 2051444751 @default.
• W2051444751 citedByCount "37" @default.
• W2051444751 countsByYear W20514447512014 @default.
• W2051444751 countsByYear W20514447512015 @default.
• W2051444751 countsByYear W20514447512016 @default.
• W2051444751 countsByYear W20514447512017 @default.
• W2051444751 countsByYear W20514447512018 @default.
• W2051444751 countsByYear W20514447512019 @default.
• W2051444751 countsByYear W20514447512020 @default.
• W2051444751 countsByYear W20514447512022 @default.
• W2051444751 countsByYear W20514447512023 @default.
• W2051444751 crossrefType "journal-article" @default.
• W2051444751 hasAuthorship W2051444751A5064165292 @default.
• W2051444751 hasAuthorship W2051444751A5064451780 @default.
• W2051444751 hasBestOaLocation W20514447511 @default.
• W2051444751 hasConcept C104317684 @default.
• W2051444751 hasConcept C134935766 @default.
• W2051444751 hasConcept C150777479 @default.
• W2051444751 hasConcept C153911025 @default.
• W2051444751 hasConcept C156719811 @default.
• W2051444751 hasConcept C175114707 @default.
• W2051444751 hasConcept C185592680 @default.
• W2051444751 hasConcept C31254050 @default.
• W2051444751 hasConcept C49105822 @default.
• W2051444751 hasConcept C552990157 @default.
• W2051444751 hasConcept C55493867 @default.
• W2051444751 hasConcept C70721500 @default.
• W2051444751 hasConcept C86803240 @default.
• W2051444751 hasConceptScore W2051444751C104317684 @default.
• W2051444751 hasConceptScore W2051444751C134935766 @default.
• W2051444751 hasConceptScore W2051444751C150777479 @default.
• W2051444751 hasConceptScore W2051444751C153911025 @default.
• W2051444751 hasConceptScore W2051444751C156719811 @default.
• W2051444751 hasConceptScore W2051444751C175114707 @default.
• W2051444751 hasConceptScore W2051444751C185592680 @default.
• W2051444751 hasConceptScore W2051444751C31254050 @default.
• W2051444751 hasConceptScore W2051444751C49105822 @default.
• W2051444751 hasConceptScore W2051444751C552990157 @default.
• W2051444751 hasConceptScore W2051444751C55493867 @default.
• W2051444751 hasConceptScore W2051444751C70721500 @default.
• W2051444751 hasConceptScore W2051444751C86803240 @default.
• W2051444751 hasIssue "14" @default.
• W2051444751 hasLocation W20514447511 @default.
• W2051444751 hasOpenAccess W2051444751 @default.
• W2051444751 hasPrimaryLocation W20514447511 @default.
• W2051444751 hasRelatedWork W134136924 @default.
• W2051444751 hasRelatedWork W184467295 @default.
• W2051444751 hasRelatedWork W1965912307 @default.
• W2051444751 hasRelatedWork W197272473 @default.
• W2051444751 hasRelatedWork W2031558647 @default.
• W2051444751 hasRelatedWork W2053929263 @default.
• W2051444751 hasRelatedWork W2075850988 @default.
• W2051444751 hasRelatedWork W2091591103 @default.
• W2051444751 hasRelatedWork W2101896454 @default.
• W2051444751 hasRelatedWork W36101119 @default.
• W2051444751 hasVolume "273" @default.
• W2051444751 isParatext "false" @default.
• W2051444751 isRetracted "false" @default.
• W2051444751 magId "2051444751" @default.
• W2051444751 workType "article" @default.