High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors
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Curated by eLife
Evaluation Summary:
This manuscript will be of interest for researchers interested in DNA repair and transcriptional regulation. The authors provide a series of well-executed and designed high-resolution sequencing data demonstrating that transcription factor (TF) binding perturbs alkylation base damage formation as well as inhibits its repair via base excision repair (BER) at TF binding sites. Moreover, they demonstrate differences between nucleotide excision repair and BER at TF binding sites that are consistent with the different repair mechanism of these two pathways. These results should have an important and timely impact on the field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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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.
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Evaluation Summary:
This manuscript will be of interest for researchers interested in DNA repair and transcriptional regulation. The authors provide a series of well-executed and designed high-resolution sequencing data demonstrating that transcription factor (TF) binding perturbs alkylation base damage formation as well as inhibits its repair via base excision repair (BER) at TF binding sites. Moreover, they demonstrate differences between nucleotide excision repair and BER at TF binding sites that are consistent with the different repair mechanism of these two pathways. These results should have an important and timely impact on the field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and …
Evaluation Summary:
This manuscript will be of interest for researchers interested in DNA repair and transcriptional regulation. The authors provide a series of well-executed and designed high-resolution sequencing data demonstrating that transcription factor (TF) binding perturbs alkylation base damage formation as well as inhibits its repair via base excision repair (BER) at TF binding sites. Moreover, they demonstrate differences between nucleotide excision repair and BER at TF binding sites that are consistent with the different repair mechanism of these two pathways. These results should have an important and timely impact on the field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Joint Public Review:
One of the most important questions in the field of genome stability is the heterogeneity of damage and repair kinetics for specific types of lesions within the genome due to chromatin structure and transcription factor binding sites. To this end several groups have begun to develop genome wide approaches to follow DNA damage and repair. This current study by an excellent team follows up an initial study published by this group in (Mao et al, Genome Res in 2017), which developed and validated methods for analyzing 7meG and 3meA formation and repair from the yeast genome after the alkylating agent methylmethane sulfonate (MMS). This original study showed that nucleosome free regions were repaired more rapidly than nucleosome containing regions. After looking at the distribution of lesions after one dose of MMS the …
Joint Public Review:
One of the most important questions in the field of genome stability is the heterogeneity of damage and repair kinetics for specific types of lesions within the genome due to chromatin structure and transcription factor binding sites. To this end several groups have begun to develop genome wide approaches to follow DNA damage and repair. This current study by an excellent team follows up an initial study published by this group in (Mao et al, Genome Res in 2017), which developed and validated methods for analyzing 7meG and 3meA formation and repair from the yeast genome after the alkylating agent methylmethane sulfonate (MMS). This original study showed that nucleosome free regions were repaired more rapidly than nucleosome containing regions. After looking at the distribution of lesions after one dose of MMS the authors studied the relatively levels of 7meG at the two transcription factors, Abf1 and Ref1 binding sites across the genome from cells versus naked DNA treatment. The authors note a 40% and 70% reduction in 7meG lesions using a 5 bp sliding window in Abf1 and Ref1, respectively. They also note that the high occupancy Ref1 sites showed this decrease in 7meG lesion formation, whereas the low occupancy sites did not. However, high resolution mapping of these two binding sites indicated as much as a 1.5 increase at specific positions within the binding sites, and Reb1A showed an increase in 3meA at a -3 position. The kinetics of repair were examined at 1 hr and 2 hr in both WT and Mag1 deficient strain. The authors confirm and validate their findings using two additional genomic approaches ORGANIC and ChIP-exo and an additional TF binding protein, Rap1. Using a robust approach involving anchor-away strains which rapidly deplete the levels of Abf1 and Reb1, indicated that the differences in the MMS-induced lesions noted in these binding sites were lost. Repair kinetic analysis suggest that removal of 7meG and 3meA, is suppressed within the Abf1 and Reb1 binding sites. High resolution analysis suggests that the +4 position of the Reb1 high occupancy binding site is refractory to repair. In order to test this hypothesis, the authors show in a biochemical assay that a substrate containing inosine is poorly removed by Mag1/Ape1 when bound by Ref1. In the final set of experiments the authors next nicely demonstrate that removal of UV-induced CPD is also influenced by TF binding extending further beyond the influence observed for alkylation repair.
The results presented in this study provide an important new information on the effects of two transcription factors on formation and repair of damage at hundreds of binding sites within the yeast genome. The results may contribute to explaining why cancer mutations are frequently elevated at TF binding sites. The manuscript is well-written, however, the way some of the experimental results were split in the main text and supplemental figures made the overall manuscript a bit cumbersome to work through and the authors miss an opportunity for a nice summary figure showing high resolution mapping of lesions and repair kinetics within the Reb1 binding sites. The data analysis would benefit from rigorous statistical analysis.
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