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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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Poly(ADP-ribose) binding and macroH2A mediate recruitment and functions of KDM5A at DNA lesions
The histone demethylase KDM5A erases histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDRs). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a noncanonical poly(ADP-ribose) (PAR)–binding region unique to KDM5A. Loss of the PAR-binding region or treatment with PAR polymerase (PARP) inhibitors (PARPi’s) blocks KDM5A–PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and function at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR binding and macroH2A1.2 in KDM5A recognition of DNA lesion sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrity.
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- Award ID(s):
- 1715370
- PAR ID:
- 10229080
- Publisher / Repository:
- DOI PREFIX: 10.1083
- Date Published:
- Journal Name:
- Journal of Cell Biology
- Volume:
- 220
- Issue:
- 7
- ISSN:
- 0021-9525
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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