University of Ottawa Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada; Agriculture and Agri-Food Canada, 2585 County Road 20, Harrow, Ontario N0R 1G0, Canada.
University of Ottawa Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
Mutat Res. 2023 Jan-Jun;826:111814. doi: 10.1016/j.mrfmmm.2023.111814. Epub 2023 Jan 6.
Mutagenesis can be thought of as random, in the sense that the occurrence of each mutational event cannot be predicted with precision in space or time. However, when sufficiently large numbers of mutations are analyzed, recurrent patterns of base changes called mutational signatures can be identified. To date, some 60 single base substitution or SBS signatures have been derived from analysis of cancer genomics data. We recently reported that the ubiquitous signature SBS5 matches the pattern of single nucleotide polymorphisms (SNPs) in humans and has analogs in many species. Using a temperature-sensitive single-stranded DNA (ssDNA) mutation reporter system, we also showed that a similar mutational pattern in yeast is dependent on error-prone translesion DNA synthesis (TLS) and glycolytic sugar metabolism. Here, we further investigated mechanisms that are responsible for this form of mutagenesis in yeast. We first confirmed that excess sugar metabolism leads to increased mutation rate, which was detectable by fluctuation assay. Since glycolysis is known to produce excess protons, we then investigated the effects of experimental manipulations on pH and mutagenesis. We hypothesized that yeast metabolizing 8% glucose would produce more excess protons than cells metabolizing 2% glucose. Consistent with this, cells metabolizing 8% glucose had lower intracellular and extracellular pH values. Similarly, deletion of vma3 (encoding a vacuolar H-ATPase subunit) increased mutagenesis. We also found that treating cells with edelfosine (which renders membranes more permeable, including to protons) or culturing in low pH media increased mutagenesis. Analysis of the mutational pattern attributable to 20 µM edelfosine treatment revealed similarity to the SBS5-like TLS- and glycolysis-dependant mutational patterns previously observed in ssDNA. Altogether, our results agree with multiple biochemical studies showing that protonation of nitrogenous bases can alter base pairing so as to stabilize some mispairs, and shed new light on a common form of intrinsic mutagenesis.
突变可以被认为是随机的，因为每个突变事件的发生在空间或时间上都无法精确预测。然而，当分析足够数量的突变时，可以识别出称为突变特征的碱基变化的反复出现模式。迄今为止，已经从癌症基因组学数据的分析中得出了大约 60 个单碱基替换或 SBS 特征。我们最近报告说，普遍存在的特征 SBS5 与人类单核苷酸多态性 (SNP) 的模式相匹配，并且在许多物种中都有类似物。使用温度敏感的单链 DNA (ssDNA) 突变报告系统，我们还表明，酵母中类似的突变模式依赖于易错跨损伤 DNA 合成 (TLS) 和糖酵解糖代谢。在这里，我们进一步研究了导致酵母中这种形式突变的机制。我们首先证实，过量的糖代谢会导致突变率增加，这可以通过波动测定法检测到。由于糖酵解已知会产生过量的质子，我们随后研究了实验操作对 pH 值和突变的影响。我们假设，代谢 8%葡萄糖的酵母会产生比代谢 2%葡萄糖的酵母更多的多余质子。与这一假设一致的是，代谢 8%葡萄糖的细胞具有较低的细胞内和细胞外 pH 值。同样，vma3（编码液泡 H+-ATPase 亚基）的缺失会增加突变。我们还发现，用埃德尔福辛（使膜更具渗透性，包括对质子）处理细胞或在低 pH 培养基中培养会增加突变。对 20µM 埃德尔福辛处理归因的突变模式的分析显示与之前在 ssDNA 中观察到的 SBS5 样 TLS 和糖酵解依赖的突变模式相似。总的来说，我们的结果与多项生化研究一致，这些研究表明，氮碱基的质子化可以改变碱基配对，从而稳定一些错配，并为一种常见的内在突变形式提供了新的认识。
