Mechanisms of glycosylase induced genomic instability

Abstract

Human alkyladenine DNA glycosylase (AAG) initiates base excision repair (BER) to guard against mutations by excising alkylated and deaminated purines. Counterintuitively, increased expression of AAG has been implicated in increased rates of spontaneous mutation in microsatellite repeats. This microsatellite mutator phenotype is consistent with a model in which AAG excises bulged (unpaired) bases, altering repeat length. To directly test the role of base excision in AAG-induced mutagenesis, we conducted mutation accumulation experiments in yeast overexpressing different variants of AAG and detected mutations via high-depth genome resequencing. We also developed a new software tool, hp_caller, to perform accurate genotyping at homopolymeric repeat loci. Overexpression of wild-type AAG elevated indel mutations in homopolymeric sequences distributed throughout the genome. However, catalytically inactive variants (E125Q/E125A) caused equal or greater increases in frameshift mutations. These results disprove the hypothesis that base excision is the key step in mutagenesis by overexpressed wild-type AAG. Instead, our results provide additional support for the previously published model wherein overexpressed AAG interferes with the mismatch repair (MMR) pathway. In addition to the above results, we observed a dramatic mutator phenotype for N169S AAG, which has increased rates of excision of undamaged purines. This mutant caused a 10-fold increase in point mutations at G:C base pairs and a 50-fold increase in frameshifts in A:T homopolymers. These results demonstrate that it is necessary to consider the relative activities and abundance of many DNA replication and repair proteins when considering mutator phenotypes, as they are relevant to the development of cancer and its resistance to treatment.

Biomarker-guided implementation of the old drug temozolomide as a novel treatment option for patients with metastatic colorectal cancer

“…BER-initiating enzyme APNG might be involved in temozolomide resistance due to increase in repair of N3 and N7 adducts in clinical setting [61]. Increased expression of APNG has been linked to genomic instability due to increase in spontaneous mutations, especially frameshift ones [85,86]. Overall, these data suggest that acquired resistance to temozolomide might develop independently from the induction of MGMT expression and may include MMR inactivation and/or acquisition of a mutator phenotype (Fig.…”

Genetic and chemotherapeutic influences on germline hypermutation

“…3a ). MPG has not yet been described as a cancer-susceptibility gene, but studies in yeast and mice have demonstrated variants in this gene and, specifically, in its substrate-binding pocket, can lead to a mutator phenotype 47 , 48 (Supplementary Table 6 ). We examined the functional impact of the observed A135T variant using in vitro assays ( Methods and Extended Data Figs.…”

Genetic and chemotherapeutic causes of germline hypermutation

“…Protein concentration was determined by absorbance at 280 nm. Active concentration was determined by electrophoretic mobility shift assay with 5'FAM-labeled pyrolidine-DNA (Supplemental Figure 8) 46 . Glycosylase assays were performed with 50 mM NaMOPS, pH 7.3, 172 mM potassium acetate, 1 mM DTT, 1 mM EDTA, 0.1 mg/mL BSA at 37 o C. For single turnover glycosylase activity, a 5'-FAM-labeled duplex was annealed by heating to 95 o C and slowly cooling to 4 o C (see Supplemental Figure 9).…”

“…In the context of the protein, the variant amino-acid forms part of the substrate binding pocket and likely affects substrate specificity (Figure 3a). MPG has not yet been described as a cancer susceptibility gene, but studies in yeast and mice have demonstrated variants in this gene, and specifically the substrate binding pocket, can lead to a mutator phenotype 47,48 . We explored the functional impact of the observed A135T variant using in vitro assays (Methods, Supplemental Figures 8, 9).…”

“…The maximal rate of excision is increased by 2-fold for 𝜺A which is among the largest increases that have been observed for 15 reported MPG variants (Supplemental Table 4). Another variant, N169S, which also shows an increase in N-glycosidic bond cleavage with the 𝜺A substrate has been established as a mutator in yeast 48,49 . These assays confirm that the A135T substitution alters the MPG binding pocket and changes the activity towards different DNA adducts.…”