Abstract
DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N-methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data show that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs ARS binding factor 1 (Abf1) and rDNA enhancer binding protein 1 (Reb1), but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. Repair of ultraviolet (UV) damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base 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.
Highlights
The interplay between base damage formation and base excision repair (BER) may play an important role in affecting mutation frequency in gene regulatory regions. 40 Introduction 41 DNA in living cells is exposed to an array of genotoxic agents, both endogenous and exogenous
NMP-seq is a sequencing method developed to map 7meG and 3meA lesions across the genome (Mao et al, 2017). This method employs BER enzymes alkyladenine-DNA glycosylase (AAG) and APE1 to digest methyl methanesulfonate (MMS)-damaged DNA and create a nick at the NMP lesion site, which is ligated to adaptor DNA for next-generation sequencing
Using a UV damage mapping method cyclobutane pyrimidine dimer sequencing (CPD-seq), we previously showed that formation of UV-induced CPDs is significantly suppressed at ARS binding factor 1 (Abf1) and rDNA enhancer binding protein 1 (Reb1) binding sites (Mao et al, 2016)
Summary
While TFs mainly function in transcriptional regulation, their binding to DNA can affect DNA damage formation and repair (Mao and Wyrick, 2019). To this end, several TF proteins have been shown to modulate formation of ultraviolet (UV) light-induced photolesions (Frigola et al, 2021; Hu et al, 2017; Mao et al, 2018) and inhibit nucleotide excision repair (NER) (Conconi et al, 1999; Sabarinathan et al, 2016). The altered UV damage formation and suppressed NER are believed to cause increased mutation rates at TF binding sites in skin cancers (Frigola et al, 2021; Mao et al, 2018; Sabarinathan et al, 2016). We used NMP-seq to analyze alkylation damage formation and BER at the binding sites of ARS binding factor 1 (Abf1) and rDNA enhancer binding protein 1 (Reb1), two essential yeast TFs that have been extensively characterized. The genome-wide binding sites for Abf and Reb have been identified at near base-pair resolution (Kasinathan et al, 2014; Rossi et al, 2021) and the DNA-binding mechanisms were analyzed in previous studies (Jaiswal et al, 2016; McBroom and Sadowski, 1994a)
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