Analysis of alkylation damage formation and base excision repair at yeast transcription factor binding sites

DNA base damage arises frequently in all living cells and is an important contributor to mutations and genome instability. The main repair pathway for base damage is base excision repair (BER). How the formation and repair of base lesions are modulated by DNA-binding proteins is poorly understood. Here we used a high-throughput damage mapping method, N-methylpurine-sequencing (NMP-seq), to characterize alkylation damage distribution and BER at yeast transcription factor (TF) binding sites upon the treatment with alkylating agent methyl methanesulfonate (MMS). We found that formation of alkylation damage was mainly suppressed at the binding sites of yeat TFs Abf1 and Reb1, but individual hotspots with elevated damage formation were also observed. Furthermore, our data indicates that repair of alkyhlation damage by BER was significantly inhibited both within the TF core motif and its adajcent DNA. The modulation of damage formation and BER was caused by the TF binding, because lesion formation and repair can be restored by depletion of Abf1 or Reb1 from the nucleus. Finally, we show that repair of UV damage by nucleotide excision repair (NER) was also inhibited at the binding sites of Abf1 and Reb1. A comparision between alkylyation and UV damage repair reveals that NER was inhibited in a broader DNA region relative to BER. Thus, our analyses indicate that TF binding significantly modulates alkylation damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.DNA碱基损伤在所有活细胞中均频繁发生，是引发突变与基因组不稳定的重要诱因。碱基损伤的主要修复通路为碱基切除修复（base excision repair, BER）。目前学界对DNA结合蛋白如何调控碱基损伤的形成与修复机制仍知之甚少。本研究采用高通量损伤定位方法——N-甲基嘌呤测序（N-methylpurine-sequencing, NMP-seq），对经烷化剂甲基磺酸甲酯（methyl methanesulfonate, MMS）处理的酵母转录因子（transcription factor, TF）结合位点处的烷化损伤分布与碱基切除修复过程进行了表征。研究发现，烷化损伤的形成在酵母转录因子Abf1与Reb1的结合位点处整体受到抑制，但同时也观测到少量损伤形成升高的热点区域。此外，本研究数据显示，碱基切除修复对烷化损伤的修复效率在转录因子核心基序及其侧翼DNA区域中均受到显著抑制。上述损伤形成与碱基切除修复的调控效应均由转录因子结合所介导：通过敲除细胞核内的Abf1或Reb1，可使损伤形成与修复过程恢复至正常水平。最后，本研究证实，核苷酸切除修复（nucleotide excision repair, NER）对紫外损伤的修复过程在Abf1与Reb1的结合位点处同样受到抑制。对比烷化损伤与紫外损伤的修复过程可见，相较于碱基切除修复，核苷酸切除修复的抑制区域覆盖了更广泛的DNA范围。综上，本研究分析表明，转录因子结合可显著调控烷化损伤的形成，并抑制碱基切除修复通路介导的损伤修复过程。碱基损伤形成与碱基切除修复之间的相互作用，可能对基因调控区域的突变频率具有重要调控作用。