Mechanisms of glycosylase induced genomic instability

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.

人类烷基腺嘌呤DNA糖苷酶（alkyladenine DNA glycosylase, AAG）通过切除烷基化和脱氨基嘌呤启动碱基切除修复（base excision repair, BER），以此抵御突变发生。与直觉相悖的是，AAG表达上调被证实与微卫星重复序列（microsatellite repeats）的自发突变率升高相关。该微卫星突变表型符合下述模型：AAG可切除凸起（未配对）碱基，进而改变重复序列长度。为直接验证碱基切除在AAG诱导突变中的作用，我们在过表达不同AAG变体的酵母中开展了突变积累实验，并通过高深度基因组重测序检测突变位点。我们还开发了一款全新软件工具hp_caller，用于在同源多聚体重复位点（homopolymeric repeat loci）实现精准基因分型。野生型AAG的过表达会提升全基因组分布的同源多聚体序列中的插入缺失突变（indel mutations）水平。然而，催化失活变体（E125Q/E125A）引发的移码突变（frameshift mutations）增幅甚至相当或更高。上述结果推翻了"过表达野生型AAG的诱变作用关键步骤为碱基切除"这一假说。与之相反，我们的结果进一步支持了此前发表的模型：过表达的AAG会干扰错配修复（mismatch repair, MMR）通路。除上述结果外，我们还观察到N169S突变型AAG展现出显著的突变表型——该变体对未损伤嘌呤的切除速率升高。该突变体可使G:C碱基对的点突变（point mutations）增加10倍，并使A:T同源多聚体（A:T homopolymers）的移码突变升高50倍。上述结果表明，在研究与癌症发生及治疗抗性相关的突变表型时，需综合考量众多DNA复制与修复蛋白的相对活性与丰度。