Grøsvik Kristin, Tesfahun Almaz Nigatu, Muruzábal-Lecumberri Izaskun, Haugland Gyri Teien, Leiros Ingar, Ruoff Peter, Kvaløy Jan Terje, Knævelsrud Ingeborg, Ånensen Hilde, Alexeeva Marina, Sato Kousuke, Matsuda Akira, Alseth Ingrun, Klungland Arne, Bjelland Svein
Department of Chemistry, Bioscience and Environmental Technology, Centre for Organelle Research, Faculty of Science and Technology, University of Stavanger, Stavanger, Norway.
Department of Biological Sciences, University of Bergen, Bergen, Norway.
Front Microbiol. 2020 Feb 25;11:263. doi: 10.3389/fmicb.2020.00263. eCollection 2020.
The cellular methyl donor -adenosylmethionine (SAM) and other endo/exogenous agents methylate DNA bases non-enzymatically into products interfering with replication and transcription. An important product is 3-methyladenine (mA), which in is removed by mA-DNA glycosylase I (Tag) and II (AlkA). The gene is constitutively expressed, while is induced by sub-lethal concentrations of methylating agents. We previously found that AlkA exhibits activity for the reactive oxygen-induced thymine (T) lesion 5-formyluracil (fU) . Here, we provide evidence for AlkA involvement in the repair of oxidized bases by showing that the adenine (A) ⋅ T → guanine (G) ⋅ cytosine (C) mutation rate increased 10-fold in wild-type and cells exposed to 0.1 mM 5-formyl-2'-deoxyuridine (fdU) compared to a wild-type specific reduction of the mutation rate at 0.2 mM fdU, which correlated with gene induction. G⋅C → A⋅T alleviation occurred without induction (at 0.1 mM fdU), correlating with a much higher AlkA efficiency for fU opposite to G than for that to A. The common keto form of fU is the AlkA substrate. Mispairing with G by ionized fU is favored by its exclusion from the AlkA active site.
细胞内甲基供体——腺苷甲硫氨酸（SAM）以及其他内源性/外源性因子可将DNA碱基非酶促甲基化，生成干扰复制和转录的产物。一种重要的产物是3-甲基腺嘌呤（mA），它可被mA-DNA糖基化酶I（Tag）和II（AlkA）去除。Tag基因持续表达，而AlkA基因则由亚致死浓度的甲基化剂诱导表达。我们之前发现，AlkA对活性氧诱导的胸腺嘧啶（T）损伤产物5-甲酰基尿嘧啶（fU）具有活性。在此，我们通过以下实验提供证据表明AlkA参与氧化碱基的修复：与野生型细胞在0.2 mM 5-甲酰基-2'-脱氧尿苷（fdU）处理下突变率特异性降低相比，野生型和AlkA基因敲除细胞在暴露于0.1 mM fdU时，腺嘌呤（A）·胸腺嘧啶（T）→鸟嘌呤（G）·胞嘧啶（C）的突变率增加了10倍，这与AlkA基因的诱导相关。在未诱导AlkA基因（0.1 mM fdU处理）的情况下发生了G·C→A·T突变的减轻，这与AlkA对与G错配的fU的修复效率远高于对与A错配的fU的修复效率相关。fU的常见酮式是AlkA的底物。离子化的fU与G错配因其被排除在AlkA活性位点之外而受到青睐。
