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

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE183622 | GEO | 2021/12/31

REPOSITORIES: GEO

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Project description:Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) and sequencing is widely used for genome-wide profiling of protein binding, but is limited by low resolution and poor specificity and sensitivity. We have implemented a simple genome-wide ChIP protocol that starts with micrococcal nuclease-digested uncross-linked chromatin followed by affinity purification and paired-end sequencing without size-selection. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of the budding yeast TFs Abf1 and Reb1 achieved near-perfect accuracy, in contrast to other profiling methods, which were much less sensitive and specific. Unlike profiles produced using X-ChIP methods such as ChIP-exo, ORGANIC profiles are not biased toward identifying sites in accessible chromatin and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila GAGA Factor and Pipsqueak. Taken together, these results suggest that ORGANIC profiling outperforms current X-ChIP methodologies for genome-wide profiling of TF binding sites. Chromatin immunoprecipitation of micrococcal nuclease-digested native chromatin followed by paired-end sequencing (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin 'ORGANIC' profiling) of DNA-binding proteins Abf1 and Reb1 from S. cerevisiae and GAGA-binding factor (GAF) and Pipsqueak (Psq) from D. melanogaster S2 cells; and, Sono-seq (paired-end sequencing of formaldehyde cross-linked and sonicated chromatin) of yeast nuclei. Reb1 ORGANIC profiling was performed at three different salt (NaCl) concentrations (80, 150, and 600 mM) and Abf1 ORGANIC profiling was done at two different salt concentrations (80 and 600 mM) to achieve varying levels of stringency. GAF and Psq ORGANIC profiles were determined at 80 mM salt. Two replicates each of Reb1 and Abf1 600 mM ORGANIC experiments, mixed Drosophila S2 cell and S. cerevisiae nuclei Reb1 ORGANIC experiments, yeast Sono-seq, and GAF and Psq ORGANIC experiments were performed. Each S. cerevisiae and mixed S2 cell/yeast ORGANIC profiling experiment included separately sequenced input chromatin and ChIP samples. Total of 24 samples.

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

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