Recognition and processing of a new repertoire of DNA substrates by human 3-methyladenine DNA glycosylase (AAG)

Abstract

The human 3-methyladenine DNA glycosylase (AAG) recognizes and excises a broad range of purines damaged by alkylation and oxidative damage, including 3-methyladenine, 7-methylguanine, hypoxanthine (Hx), and 1,N(6)-ethenoadenine (epsilonA). The crystal structures of AAG bound to epsilonA have provided insights into the structural basis for substrate recognition, base excision, and exclusion of normal purines and pyrimidines from its substrate recognition pocket. In this study, we explore the substrate specificity of full-length and truncated Delta80AAG on a library of oligonucleotides containing structurally diverse base modifications. Substrate binding and base excision kinetics of AAG with 13 damaged oligonucleotides were examined. We found that AAG bound to a wide variety of purine and pyrimidine lesions but excised only a few of them. Single-turnover excision kinetics showed that in addition to the well-known epsilonA and Hx substrates, 1-methylguanine (m1G) was also excised efficiently by AAG. Thus, along with epsilonA and ethanoadenine (EA), m1G is another substrate that is shared between AAG and the direct repair protein AlkB. In addition, we found that both the full-length and truncated AAG excised 1,N(2)-ethenoguanine (1,N(2)-epsilonG), albeit weakly, from duplex DNA. Uracil was excised from both single- and double-stranded DNA, but only by full-length AAG, indicating that the N-terminus of AAG may influence glycosylase activity for some substrates. Although AAG has been primarily shown to act on double-stranded DNA, AAG excised both epsilonA and Hx from single-stranded DNA, suggesting the possible significance of repair of these frequent lesions in single-stranded DNA transiently generated during replication and transcription.

Figure 1

Structure of the AAG active site (green) showing the flipped-out εA nucleotide (gold). The dashed line indicates a hydrogen bond between the N6 of εA (acceptor) and the peptide amide (donor) of His136. This figure is generated using the coordinates of the AAG/εA crystal structure (PDB ID: 1f4r (9)) using Pymol.

Figure 2

Chemical structures and sequence context of the different DNA lesions tested in the present study.

Figure 3

Δ80AAG binds to different lesions with different affinities. Gel mobility shift assay for the binding of Δ80AAG to oligonucleotides containing (A) m1G, (C) e3U, (E) εA. Graphical representation of the binding of Δ80AAG to (B) m1G, (D) e3U, (F) εA.

Figure 4

m1G and EA, known AlkB substrates, are cleaved by AAG when present in double-stranded DNA. Glycosylase activity of AAG toward (A) m1G and (C) EA. No AAG represents incubation without AAG for the longest time point of the assay. Graphical representation of the glycosylase activity toward (B) m1G and (D) EA by (■) Δ80AAG and (▲) full-length AAG. For comparison with EA, the data of AAG glycosylase activity towards εA from Figure 5B is also shown in (D): (●) Δ80AAG and () full-length AAG.

Figure 5

AAG cleaves εA and Hx in both double-stranded and single-stranded DNA. Glycosylase activity of AAG toward εA and Hx. Gels showing AAG excision of εA in (A) double- and single-stranded DNA. No AAG represents incubation without AAG for the longest time point of the assay. Graphical representation of the glycosylase activity toward εA in (B) duplex DNA and (C) singlestranded DNA by (■) Δ80AAG and (▲) full-length AAG. Gels showing excision of Hx in (D) double- and single-stranded DNA. No AAG represents incubation without AAG for the longest time point of the assay. Graphical representation of the glycosylase activity toward Hx in (E) duplex and (F) single-stranded DNA by (■) Δ80AAG and (▲) full-length AAG.

Figure 6

1,N²-εG in double-stranded DNA is a substrate for both the Δ80 truncated form and the full-length AAG protein. (A) Glycosylase activity of AAG toward 1,N²-εG by Δ80 and full-length AAG. No AAG represents incubation without AAG for the longest time point of the assay. (B) Graphical representation of the glycosylase activity toward 1,N²-εG by (■) Δ80AAG and (▲) full-length AAG.

Figure 7

Uracil is a substrate for the full-length AAG protein in both single- and double-stranded DNA. (A) Glycosylase activity of Δ80 vs. full-length AAG toward U. (B) Glycosylase activity of full-length AAG toward U. No AAG represents incubation without AAG for the longest time point of the assay. (C) Graphical representation of the glycosylase activity toward U by full-length AAG in (■) single-stranded and (▲) double-stranded DNA.