Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG

Abstract

The human 3-methyladenine DNA glycosylase [alkyladenine DNA glycosylase (AAG)] catalyzes the first step of base excision repair by cleaving damaged bases from DNA. Unlike other DNA glycosylases that are specific for a particular type of damaged base, AAG excises a chemically diverse selection of substrate bases damaged by alkylation or deamination. The 2.1-Å crystal structure of AAG complexed to DNA containing 1,N 6 -ethenoadenine suggests how modified bases can be distinguished from normal DNA bases in the enzyme active site. Mutational analyses of residues contacting the alkylated base in the crystal structures suggest that the shape of the damaged base, its hydrogen-bonding characteristics, and its aromaticity all contribute to the selective recognition of damage by AAG. DNA bases are chemically reactive and readily undergo deamination and alkylation on the inevitable exposure to reactive cellular metabolites and environmental toxicants (1-4). Alkylation occurs at many different positions of DNA, producing a variety of lesioned bases (4, 5) that can block replication or interfere with other enzymatic activities templated by DNA. Hypoxanthine is an abundant deaminated base, and it too corrupts the DNA template. Remarkably, human cells appear to produce a single enzyme, alkyladenine DNA glycosylase [AAG (3-methyladenine DNA glycosylase, ANPG, or MPG)], which recognizes and removes hypoxanthine plus a variety of alkylated bases that include 3-methyladenine, 7-methylguanine, and 1,N 6 -ethenoadenine (A; refs. 6 -13). AAG cleaves the N-glycosylic bond joining the damaged base to the DNA backbone, and the resulting abasic nucleotide is excised and replaced with a normal nucleotide by the sequential action of an endonuclease, a polymerase, and DNA ligase (14). The high selectivity for damaged vs. normal bases is essential because normal bases are present in vast excess. AAG can distinguish alternations in both adenine and guanine and can recognize changes present in both the major and minor grooves of DNA. We set out to determine how AAG achieves selectivity for chemically diverse substrates.We previously reported a 2.7-Å crystal structure of AAG complexed to DNA containing a transition-state mimic of the glycosylase reaction, the pyrrolidine abasic nucleotide (pyr; PDB ID code 1bnk; refs. 15 and 16). In the AAG͞pyr-DNA complex, the pyr ring is f lipped into the proposed active site by intercalation of the Tyr-162 side chain into the minor groove of the DNA (15). A bound water molecule in the active site is aligned for a back-side attack of the abasic sugar, but the pyr inhibitor lacks a base, and we could not deduce how AAG recognizes alkylated bases in preference to normal bases. Structures of several other DNA N-glycosylases complexed to their DNA substrates have been reported (17)(18)(19)(20). These enzymes are selective for one type of damaged DNA base and, correspondingly, their active site structures are tailor made for specific interactions with these substrates. For example, uracil DNA glycosylase f lips ur...

Altering DNA base excision repair: Use of nuclear and mitochondrial‐targeted N‐methylpurine DNA glycosylase to sensitize astroglia to chemotherapeutic agents

“…1C). Although the E125A MPG has been shown to possess minimal glycosylase activity (Lau et al, 2000), the in vitro assays also revealed a reduced level of MPG activity in mitochondrial lysates taken from MutMitoMPG astrocytes. These findings indicate that adenoviral delivery is an effective means of modulating MPG activity in either the nucleus or mitochondria of astrocytes maintained in primary culture.…”

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene

“…For example, in yeast, when BER is imbalanced by an excess of abasic sites (generated by over-expression N-methylpurine-DNA glycosylases, MPG), the rate of spontaneous mutation is increased signi®cantly (Chakravarti et al, 1991;Berdal et al, 1998;Glassner et al, 1998;Lau et al, 2000). Chinese hamster ovary cells over-expressing human MPG (a broad spectrum glycosylase), however, when exposed to methylating agents, did not show increased mutagenesis but showed inhibition of DNA synthesis, chromosomal aberrations and DNA breaks (Ibeanu et al, 1992;Coquerelle et al, 1995).…”

Oxidative dealkylation DNA repair mediated by the mononuclear non-heme iron AlkB proteins

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