Global screening of base excision repair in nucleosome core particles.

DNA damage is a fundamental molecular cause of genomic instability. Base excision repair (BER) is one line of defense to minimize the potential mutagenicity and/or toxicity derived from damaged nucleobase lesions. However, BER in the context of chromatin, in which eukaryotic genomic DNA is compacted through a hierarchy of DNA-histone protein interactions, is not fully understood. Here, we investigate the activity of BER enzymes at 27 unique geometric locations in a nucleosome core particle (NCP), which is the minimal unit of packaging in chromatin. The BER enzymes include uracil DNA glycosylase (UDG), AP endonuclease 1 (APE1), DNA polymerase β (Pol β), and DNA ligase IIIα complexed with X-ray repair cross complementing group 1 (LigIIIα/XRCC1). This global analysis of BER reveals that initiation of the repair event by UDG is dictated by the rotational position of the lesion. APE1 has robust activity at locations where repair is initiated whereas the repair event stalls at the Pol β nucleotide incorporation step within the central ∼45 bp of nucleosomal DNA. The final step of the repair, catalyzed by LigIIIα/XRCC1, is achieved only in the entry/exit regions of the NCP when nick sites are transiently exposed by unwrapping from the histones. Kinetic assays further elucidate that the location of the damaged lesion modulates enzymatic activity. Notably, these data indicate that some of the BER enzymes can act at a significant number of locations even in the absence of chromatin remodelers or other cellular factors. These results inform genome wide maps of DNA damage and mutations and contribute to our understanding of mutational hotspots and signatures.