Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 54 (9), 1849-57

Base Excision Repair Enzymes Protect Abasic Sites in Duplex DNA From Interstrand Cross-Links

Affiliations

Base Excision Repair Enzymes Protect Abasic Sites in Duplex DNA From Interstrand Cross-Links

Suzanne J Admiraal et al. Biochemistry.

Abstract

Hydrolysis of the N-glycosyl bond between a nucleobase and deoxyribose leaves an abasic site within duplex DNA. The abasic site can react with exocyclic amines of nucleobases on the complementary strand to form interstrand DNA-DNA cross-links (ICLs). We find that several enzymes from the base excision repair (BER) pathway protect an abasic site on one strand of a DNA duplex from cross-linking with an amine on the opposing strand. Human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) accomplish this by binding tightly to the abasic site and sequestering it. AAG protects an abasic site opposite T, the product of its canonical glycosylase reaction, by a factor of ∼10-fold, as estimated from its inhibition of the reaction of an exogenous amine with the damaged DNA. Human apurinic/apyrimidinic site endonuclease 1 and E. coli endonuclease III both decrease the amount of ICL at equilibrium by generating a single-strand DNA nick at the abasic position as it is liberated from the cross-link. The reversibility of the reaction between amines and abasic sites allows BER enzymes to counter the potentially disruptive effects of this type of cross-link on DNA transactions.

Figures

Figure 1
Figure 1
Possible fates of abasic sites within duplex DNA. Abasic sites can react with an amine group of a protein (blue) to form a DPC or with an exocyclic amine of a nucleobase (yellow) to form an ICL.
Figure 2
Figure 2
Sequences of oligonucleotides used in this study. The abbreviations ab and P denote abasic and 2-aminopurine residues, respectively, and -S-(6-fam) indicates the location of a phosphorothioate linked to a fluorescein label.
Figure 3
Figure 3
AAG prevents an ICL from forming between an abasic site and P located on opposing strands of a 35mer DNA duplex. A. A 35mer-35mer ICL builds up over time in reactions that contain 0.25 µM duplex 1/3 treated with UDG, but AAG reduces the overall amount of ICL that accumulates. Reactions were carried out at 37 °C and pH 6.5, and samples were quenched and subjected to mild alkaline hydrolysis prior to separation on a 20% denaturing polyacrylamide gel. The slightly higher levels of 35mer present at the earliest time points in reactions containing UDG reveal that the conversion of intact 1/3 to abasic 2/3 is not quite complete at these times. B. The fraction of ICL at time points in A and duplicate reactions is shown. AAG reduces the amount of ICL that forms, with only trace amounts detected when the AAG concentration exceeds the DNA concentration. Lines are exponential fits to the data and give end points of 5% ICL in the absence of AAG, 2.8% ICL in the presence of 0.125 µM AAG, and 0.4% ICL in the presence of 0.5 µM AAG.
Figure 4
Figure 4
An ICL forms between an abasic site and P located on opposing strands of the 35mer DNA duplex 2/3. A. The amount of 35mer-35mer ICL increases with time in a reaction that contains duplex 2/3 (lanes 3–7). No ICL forms in control reactions containing duplex 2/4 (lane 1) or single stranded 2 (lane 2). Reactions contained 0.25 µM DNA and were carried out at 37 °C and pH 6.5. Samples were reduced with NaBH4 prior to separation on a 20% denaturing polyacrylamide gel. The 17mer that is visible in all lanes arises from nonenzymatic β-elimination of 2 that occurs during preparation and storage of this oligonucleotide, and its amount (6%) remains constant during the reaction courses. B. The ratio of ICL to unlinked 2/3 at time points in A and duplicate reactions is shown. An end point ratio of 0.047 and kobs of 0.034 min−1 for the approach to the end point were obtained from an exponential fit to the data.
Figure 5
Figure 5
The equilibrium between the species with and without an ICL for duplex 2/3. The Keq value shown is for the duplex at 37 °C and pH 6.5, from Figure 4B.
Figure 6
Figure 6
ICL breakdown due to BER enzymes. A. Duplex 2/3 ICL disappears over time in reactions containing 0.5 µM APE1 or 0.5 µM Nth; exponential fits to the data give values of kobs of 0.05 min−1 and 0.03 min−1 for the reactions containing APE1 and Nth, respectively. B. Duplex 2/3 ICL disappears with kobs= 0.03 min−1 in reactions containing either 0.5 µM AAG or 0.5 µM AlkA. For both A and B, BER enzymes were added to 0.25 µM duplex 2/3 at equilibrium at 37 °C and pH 6.5, and samples were reduced with NaBH4 prior to separation on 20% denaturing polyacrylamide gels (Figure S2). APE1 and Nth reactions included 0.25 mM MgCl2.
Figure 7
Figure 7
Reaction of PFBHA and abasic DNA. A. The oxyamine adduct PFBHA-DNA is the product of the reaction between PFBHA and abasic DNA. This reaction is analogous to the well-characterized reaction of methoxyamine with abasic DNA but has the advantage that the larger PFBHA adduct is more easily separated from the abasic DNA substrate on a gel. B. PFBHA reacts with 2 or 2/3 to form PFBHA-DNA. No adduct forms in the absence of PFBHA. Reactions contained 0.25 µM DNA and were carried out at 37 °C and pH 6.5 with or without 1.5 mM PFBHA. Samples were reduced with NaBH4 before separation on a 20% denaturing polyacrylamide gel. This image displays only the region of the gel where the 35mer and the PFBHA-35mer appear; A full image of the same gel (Figure S3) shows that ICL formation is minimal in reactions of duplex 2/3 when PFBHA is present, because PFBHA outcompetes the exocyclic amine for reaction with the abasic site at the concentrations of PFBHA used. C. The kobs for formation of PFBHA-DNA increases with PFBHA concentration in duplex 2/3 reactions. Values of kobs at each PFBHA concentration were determined from exponential fits to the data and are plotted in D. D. Linear fits for the dependence of kobs on PFBHA concentration give second order rate constants for the reaction of PFBHA with 2 of 0.06 mM−1min−1 and with 2/3 of 0.14 mM−1min−1.
Figure 8
Figure 8
AAG and AlkA protect abasic DNA in a duplex from PFBHA. Reactions of duplex 2/3 (A), single stranded 2 (B), and duplex 2/4 (C) contained 0.25 µM DNA, 1.5 mM PFBHA, and 1.5 µM AAG, 1.5 µM AlkA, or no added enzyme. Samples from reactions performed at 37 °C and pH 6.5 were reduced with NaBH4 prior to separation on 20% denaturing polyacrylamide gels. Exponential fits to the data are shown, and kobs values from the fits are recorded in Table 1.
Figure 9
Figure 9
BER enzymes prevent abasic sites in duplex DNA from cross-linking by binding tightly to the free abasic sites (AAG, AlkA) or by reacting irreversibly with them (APE1, Nth). An abasic pyrrolidine nucleotide is flipped into the active site of AAG in the structure that is shown.

Similar articles

See all similar articles

Cited by 7 articles

See all "Cited by" articles

Publication types

Feedback