Analysis of cytosine deamination events in excision repair sequencing reads reveals mechanisms of incision site selection in NER.

Nucleotide excision repair (NER) removes helix-distorting DNA lesions and is therefore critical for genome stability. During NER, DNA is unwound on either side of the lesion and excised, but the rules governing incision site selection, particularly in eukaryotic cells, are unclear. Excision repair-sequencing (XR-seq) sequences excised NER fragments, but analysis has been limited because the lesion location is unknown. Here, we exploit accelerated cytosine deamination rates in UV-induced CPD (cyclobutane pyrimidine dimer) lesions to precisely map their locations at C to T mismatches in XR-seq reads, revealing general and species-specific patterns of incision site selection during NER. Our data indicate that the 5' incision site occurs preferentially in HYV (i.e. not G; C/T; not T) sequence motifs, a pattern that can be explained by sequence preferences of the XPF-ERCC1 endonuclease. In contrast, the 3' incision site does not show strong sequence preferences, once truncated reads arising from mispriming events are excluded. Instead, the 3' incision is partially determined by the 5' incision site distance, indicating that the two incision events are coupled. Finally, our data reveal unique and coupled NER incision patterns at nucleosome boundaries. These findings reveal key principles governing NER incision site selection in eukaryotic cells.