The Action of DNA Ligase at Abasic Sites in DNA*

Daniel F. BogenhagenDagger and Kevin G. Pinz

From the Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References

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 beta -elimination mechanism. This beta -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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

DNA repair pathways have evolved to process a wide range of chemically distinct lesions in DNA (1). 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).1 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 (2). The latter residue, referred to as a 5'-dRP moiety, is relatively alkali labile and can be removed by AP lyases that facilitate beta -elimination. Most enzymes demonstrated to have dRPase activity operate through this lyase mechanism, although some, such as the Escherichia coli recJ protein, catalyze hydrolysis (3). 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 (4), 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 (5-9) and, more recently, for DNA pol beta  (10, 11).

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 (12). 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 beta -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.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- mtDNA ligase, mitochondrial AP endonuclease, and DNA ligase I were purified from Xenopus ovary tissue as described (12). 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 (13) 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. 7C to permit better resolution of the reaction product from the initial labeled substrate.

Methods-- Oligonucleotides were phosphorylated using standard procedures (14) and annealed by heating to 80 °C in 0.1 M 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 (9, 11) by including 20 or 50 mM NaBH4 in the binding reaction. After 30 min at 25 °C, the solution was adjusted to contain 6 mM CaCl2 and 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 (13), and detected by autoradiography or PhosphorImager analysis (Molecular Dynamics). dRPase activity was measured as described (3), and HPLC analysis of products generated in the presence of sodium thioglycolate was performed as described (5, 15), except that a Poros Q anion exchange column was used.

    RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

DNA Ligases Are Able to Reseal DNA Strands Nicked on the 5' Side of an AP Site-- 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 (12, 15). 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 (16) 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 (15, 17). This is an analogue of a reduced deoxyribose moiety and is not subject to beta -elimination. Experiments presented below show that T4 DNA ligase can also reseal an authentic (nonreduced) AP site.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   DNA ligases can reseal a nick adjacent to a 5'-phosphorylated abasic site. A 5'-end labeled oligonucleotide containing a single synthetic AP site (3-hydroxy-2-hydroxymethyltetrahydrofuran, designated F for furan) was ligated into a gapped heteroduplex, and the covalently closed circular DNA product was purified as described (15). PAGE-urea gel analysis of a HinfI digest of this substrate confirmed that all substrate molecules were ligated with the tetrahydrofuran residue embedded in a 46-mer fragment, denoted as 46(F) (lane 1). Treatment with mitochondrial AP endonuclease led to cleavage on the 5' side of the tetrahydrofuran residue, providing a 26-mer fragment following diagnostic HinfI cleavage (lane 2). This fragment with a 5'-tetrahydrofuran residue is identified as F26. Samples of this mitochondrial AP endonuclease-incised substrate were incubated with X. laevis DNA ligase I, mtDNA ligase, T4 DNA ligase, or T7 DNA ligase (lanes 3-6). The products were deproteinized by organic extraction and cleaved with HinfI endonuclease prior to electrophoresis. The radioactive species moving slightly slower than the F26 fragment, which is most apparent in lane 6, is an intermediate in the ligase reaction produced by transfer of AMP to the 5' terminus at the nick.

T4 DNA Ligase Has AP Lyase Activity-- We previously showed Xenopus laevis mtDNA ligase can be labeled using a borohydride trapping procedure that is specific for AP lyase activities (12). 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.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2.   Bacteriophage DNA ligases react in a borohydride trapping assay for AP lyases. An autoradiogram of an SDS-PAGE analysis of labeled DNA ligases is shown with the positions of protein molecular mass markers in kDa indicated on the left. To provide radioactively labeled markers, T4 and T7 DNA ligase were incubated with [alpha -32P]ATP (lanes 1) in a buffer containing 20 mM Tris, pH 8.0, 10 mM MgCl2, 5 mM dithiothreitol, 0.02% Triton X-100 to permit adenylation of the active site lysine. For borohydride trapping reactions, DNA ligase was incubated with a duplex oligonucleotide with an internal nick adjacent to a 5'-32P-U residue (lanes 2 and 3) or with a 5'-32P-T residue as a control (lanes 4) as diagramed at the bottom of the figure. 1 pmol of the oligonucleotide substrate was incubated with 0.1 unit of UDG and 100 ng of either T4 DNA ligase or T7 DNA ligase in 20 µl of 10 mM Hepes, pH 7.5, 1 mM MgCl2 and either 50 mM NaBH4 (lanes 2 and 4) or 50 mM NaCl (lanes 3). The reactions were treated with micrococcal nuclease and labeled proteins were detected by following SDS-PAGE as described under "Experimental Procedures."

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 mM MgCl2 and is inhibited more than 90% by 1 mM ATP (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. 3B). 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. 3B. These borohydride cross-linking results are consistent with the hypothesis that T4 DNA ligase contains AP lyase activity.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3.   Characterization of the borohydride trapping reaction for T4 DNA ligase. A, the UDG-treated nicked oligonucleotide substrate used in Fig. 2 was incubated with T4 DNA ligase in reactions containing 10 mM Hepes, pH 7.5, 50 mM NaBH4 and the indicated concentrations (in mM) of either MgCl2 or EDTA and with ATP in one reaction (lane 5). B, standard borohydride trapping reactions with T4 DNA ligase were performed, comparable with lane 2 of A. Lane 2 shows the effect of omitting post-treatment with micrococcal nuclease (MN). In both panels, the arrowheads indicate the mobility of T4 DNA ligase.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   The chemistry of AP lyase action accounts for DNA-enzyme and dRP-enzyme complexes following NaBH4 treatment. This scheme is based on those presented for other AP lyase enzymes (7, 19). 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 beta -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.

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. 5, 7, and 18).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5.   Release of an acid soluble product from a 5'-32P-labeled abasic site by T4 DNA ligase. 15 pmol of 5'-32P U17 oligonucleotide pretreated with UDG was incubated with 100 ng of T4 DNA ligase in 40 mM Hepes buffer, pH 7.5, for varied periods of time at 30 °C (A), for 30 min at varied temperature (B), or in the presence of increasing concentrations of ATP (C). dRP release was measured as the generation of a radioactive product soluble in the presence of cold TCA and activated charcoal (3). The percentage of dRP removed was determined relative to the total alkali-labile cpm. The maximal amount of label solubilized in parallel reactions without enzyme represented 4% of the total available substrate.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 6.   The 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 (5, 18). Reaction of the beta -elimination product with thioglycolate leads to reduction of the aldehyde, blocking the subsequent delta -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 1 M NaCl step.

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 beta  possesses AP lyase activity (10, 11). Although T4 DNA ligase is capable of processing multiple AP site substrates (Fig. 5A), the turnover number is less than 10% of the vigorous rate observed for FPG protein (5). It should be noted that the glycosylase activity of FPG protein is reported to be significantly slower than the AP lyase activity (5). 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 (19). The fact that the AP lyase activity is suppressed by ATP suggests that the active site lysine residue that is adenylated in DNA ligase (20) may be involved directly in the nucleophilic attack that promotes beta -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 (21). This enzyme has the added advantage that it is relatively small, with only 359 amino acid residues.

A Model for the Role of AP Lyase Associated with DNA Ligase-- 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 (12), and DNA ligase I (data not shown). Because ATP-dependent DNA ligases as a class share structural and functional features (22), 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 either E. 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 beta -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.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 7.   Model for the action of DNA ligase at AP sites. A, the proposed action of DNA ligase at an incised AP site is illustrated in the presence or the absence of ATP. DNA ligase is shown as a C-shaped enzyme with a prominent groove (21). See "Results and Discussion" for details. B, a substrate containing an internal AP site was prepared by UDG treatment of a duplex oligonucleotide with a single U residue 15 nucleotides from the 32P-labeled 5'-end as diagramed at the bottom. The oligonucleotide was incubated for 45 min at 25 °C in 40 mM Hepes, pH 7.5, either without additional enzyme (lane 1) or with either E. coli FPG protein (lane 2) or T4 DNA ligase (lane 3). Reactions were treated for 15 min with 0.1 M NaBH4 to reduce the abasic sugar residue, and products were subjected to electrophoresis on a 20% PAGE-urea gel and detected by autoradiography. C, an oligonucleotide substrate was prepared by annealing a nonradioactive 12-mer adjacent to the 5'-labeled U17 oligomer to a complementary strand as diagramed at the bottom. This duplex was treated with UDG to create a 5'-dRP residue and then incubated in 40 mM Hepes, pH 7.5, buffer alone (lane 1) or with 20 ng of T4 DNA ligase without ATP (lane 2) or with the same amount of T4 DNA ligase with 0.1 mM ATP (lane 3). All incubations were for 1 h at 25 °C. Reactions were treated for 15 min with 0.1 M NaBH4, and products were subjected to electrophoresis on a 20% PAGE-urea gel and detected by autoradiography. Markers consisted of kinase-labeled oligonucleotides identified by size on the left. The 17-mer marker is the intact U17 oligonucleotide, and the 12-mer marker is a 5'-labeled sample of the same nonradioactive 12-mer used to prepare the substrate. The diagrams at the bottom of B and C show the oligonucleotides used in these experiments with the top strands oriented 5' to 3'.

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 beta -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. 7A. 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. 7B. 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 beta -elimination but unlike FPG protein does not efficiently promote delta -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. 7C. 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 3 of Fig. 7C). 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. 7C 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by National Institute of Environmental Health Sciences Grant PO1-04068.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.

Dagger To whom correspondence should be addressed. Tel.: 516-444-3068; Fax: 516-444-3218; E-mail: dan{at}pharm.sunysb.edu.

1 The 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

  1. Friedberg, E. C., Walker, G. C., and Seide, W. (1995) DNA Repair and Mutagenesis, ASM Press, Washington, D.C.
  2. Barzilay, G., and Hickson, I. D. (1995) BioEssays 17, 713-719[Medline] [Order article via Infotrieve]
  3. Dianov, G., Sedgwick, B., Daly, G., Olsson, M., Lovett, S., and Lindahl, T. (1994) Nucleic Acids Res. 22, 993-998[Abstract]
  4. Kow, Y. W., and Wallace, S. S. (1987) Biochemistry 26, 8200-8206[Medline] [Order article via Infotrieve]
  5. Graves, R. J., Felzenszwalb, I., Laval, J., and O'Connor, T. R. (1992) J. Biol. Chem. 267, 14429-14435[Abstract/Free Full Text]
  6. Dodson, M. L., Schrock, R. D. I., and Lloyd, R. S. (1993) Biochemistry 32, 8284-8290[Medline] [Order article via Infotrieve]
  7. Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677[Abstract/Free Full Text]
  8. Sun, B., Latham, K. A., Dodson, M. L., and Lloyd, R. S. (1995) J. Biol. Chem. 270, 19501-19508[Abstract/Free Full Text]
  9. Nash, H. M., Bruner, S. D., Scharer, O. D., Kawate, T., Addona, T. A., Spooner, E., Lane, W. S., and Verdine, G. L. (1996) Curr. Biol. 6, 968-980[Medline] [Order article via Infotrieve]
  10. Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702[Medline] [Order article via Infotrieve]
  11. Piersen, C. E., Prasad, R., Wilson, S. H., and Lloyd, R. S. (1996) J. Biol. Chem. 271, 17811-17815[Abstract/Free Full Text]
  12. Pinz, K. G., and Bogenhagen, D. F. (1998) Mol. Cell. Biol. 18, in press
  13. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  14. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  15. Matsumoto, Y., Kim, K., and Bogenhagen, D. F. (1994) Mol. Cell. Biol. 14, 6187-6197[Abstract]
  16. Goffin, C., Bailly, V., and Verly, W. G. (1987) Nucleic Acids Res. 15, 8755-8771[Abstract]
  17. Takeshita, M., Chang, C.-N., Johnson, F., Will, S., and Grollman, A. P. (1987) J. Biol. Chem. 262, 10171-10179[Abstract/Free Full Text]
  18. Bricteux-Gregoire, S., and Verly, W. G. (1989) Nucleic Acids Res. 17, 6269-6282[Abstract]
  19. Dodson, M. L., Michaels, M. L., and Lloyd, R. S. (1994) J. Biol. Chem. 269, 32709-32712[Free Full Text]
  20. Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404[Abstract]
  21. Subramanya, H. S., Doherty, A. J., Ashford, S. R., and Wigley, D. B. (1996) Cell 85, 607-615[Medline] [Order article via Infotrieve]
  22. Lindahl, T., and Barnes, D. E. (1992) Annu. Rev. Biochem. 61, 251-281[CrossRef][Medline] [Order article via Infotrieve]


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