Base Excision and DNA Binding Activities of Human Alkyladenine DNA Glycosylase Are Sensitive to the Base Paired with a Lesion

Abstract

The human alkyladenine DNA glycosylase has a broad substrate specificity, excising a structurally diverse group of damaged purines from DNA. To more clearly define the structural and mechanistic bases for substrate specificity of human alkyladenine DNA glycosylase, kinetics of excision and DNA binding activities were measured for several different damaged and undamaged purines within identical DNA sequence contexts. We found that 1,N 6 -ethenoadenine (⑀A) and hypoxanthine (Hx) were excised relatively efficiently, whereas 7,8-dihydro-8-oxoguanine, O 6 -methylguanine, adenine, and guanine were not. Single-turnover kinetics of excision of Hx and ⑀A paired with T showed that excision of Hx was about four times faster than ⑀A, whereas binding assays showed that the binding affinity was about five times greater for ⑀A than for Hx. The opposing pyrimidine base had a significant effect on the kinetics of excision and DNA binding affinity of Hx but a small effect on those for ⑀A. Surprisingly, replacing a T with a U opposite Hx dramatically reduced the excision rate by a factor of 15 and increased the affinity by a factor of 7-8. The binding affinity of human alkyladenine DNA glycosylase to a DNA product containing an abasic site was similar to that for an Hx lesion.The base excision repair pathway provides the cell with a major line of defense against damage to DNA bases by excising damaged bases and resynthesizing DNA. Base excision repair is initiated by the activity of DNA glycosylases, which function to identify and excise damaged bases. Because these enzymes recognize DNA base damage, they are key to the overall effectiveness of the pathway. Monofunctional DNA glycosylases such as human alkyladenine DNA glycosylase (hAAG) 1 excise damaged DNA bases by hydrolysis of the C1Ј-N glycosylic bond, forming a free DNA base and an abasic sugar residue. Once the damaged base is removed, other enzymes in the pathway remove the remaining sugar residue and resynthesize DNA to fill in the gap.DNA glycosylases are damage-specific; different enzymes are responsible for excising different types of damaged DNA bases. Some glycosylases such as uracil DNA glycosylase are very specific and excise only a single damaged base, uracil, in this case. Other DNA glycosylases have broader substrate specificities. For example, formamidopyrimidine DNA glycosylase (FaPy) recognizes oxidative damage to DNA bases and excises 7,8-dihydro-8-oxoguanine (8-oxoG), FaPy, and 5-hydroxycytosine. Based on both structural (for recent reviews, Refs. 1-3) and spectroscopic (4) data, DNA glycosylases are believed to "flip" damaged nucleotides out of the DNA helix and into an enzyme active site, where catalysis takes place. Given this type of flipping mechanism, it is easy to imagine how a DNA glycosylase may recognize a specific damaged DNA base through interactions in the active site that provide a "tight" fit and align the glycosylic bond for chemistry. For DNA glycosylases with broader substrate specificities, the nature of the structural interactions and me...

Oxanine DNA Glycosylase Activity from Mammalian Alkyladenine Glycosylase

“…Cleavage of T/I substrate approached completion in a 60-min incubation period (Fig. 5C), in keeping with a previous analysis (28).…”

“…Cleavage of inosine in the T/I base pair was primarily observed in the wild type spleen tissue, consistent with previous reports (22,34) that AAG is a major hypoxanthine DNA glycosylase in mammalian cells. In keeping with the preferential cleavage of the T/I base pair by purified mammalian AAG (28,33,(35)(36)(37), little C/I cleavage activity was observed in the wild type cell extract (Fig. 4B).…”

“…To rule out the possibility that the observed cleavage activities may come from E. coli expression host contamination, assays were performed with an active site mutant ⌬54-E125Q. This mutant is catalytically inactive because of the change of a proposed general base residue Glu to Gln (24,28,31,32). No DNA cleavage was observed from ⌬54-E125Q, indicating that the ODG activities are intrinsic to hAAG (Fig.…”

Hijacking of the Human Alkyl-N-purine-DNA Glycosylase by 3,N4-Ethenocytosine, a Lipid Peroxidation-induced DNA Adduct

“…Based on our data, we suggest that ANPG is able to flip out ⑀C from duplex DNA, and we speculate that it is the improper alignment of extruded adduct in the active site of ANPG that results in an inability to form a transition state intermediate structure. In support of this hypothesis, it has been demonstrated that the structure of the base pair rather than simply the damaged base plays a crucial role in base excision by ANPG (55).…”

Repair of exocyclic DNA adducts: rings of complexity

“…These activities are evolutionarily enhanced inasmuch as the mammalian glycosylases excise eA two to three orders of magnitude more efficiently than their yeast and bacterial functional homologs. (28) Both opposite base (28)(29)(30) and sequence context (30,31) can affect the eA activity of ANPGs but data from various studies differed in the magnitude of such effects. Recently, it was reported that the E. coli mismatchspecific uracil-DNA glycosylase (Mug) could remove eA but with extremely low efficiency.…”