Site-specific repair of cyclobutane pyrimidine dimers in a positioned nucleosome by photolyase and T4 endonuclease V invitro

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Abstract
Since genomic DNA is folded into nucleosomes, and DNA damage is generated all over the genome, a central question is how DNA repair enzymes access DNA lesions and how they cope with nucleosomes. To investigate this topic, we used a reconstituted nucleosome (HISAT nucleosome) as a substrate to generate DNA lesions by UV light (cyclobutane pyrimidine dimers, CPDs), and DNA photolyase and T4 endonuclease V (T4‐endoV) as repair enzymes. The HISAT nucleosome is positioned precisely and contains a long polypyrimidine region which allows one to monitor formation and repair of CPDs over three helical turns. Repair by photolyase and T4‐endoV was inefficient in nucleosomes compared with repair in naked DNA. However, both enzymes showed a pronounced site‐specific modulation of repair on the nucleosome surface. Removal of the histone tails did not substantially enhance repair efficiency nor alter the site specificity of repair. Although photolyase and T4‐endoV are different enzymes with different mechanisms, they exhibited a similar site specificity in nucleosomes. This implies that the nucleosome structure has a decisive role in DNA repair by exerting a strong constraint on damage accessibility. These findings may serve as a model for damage recognition and repair by more complex repair mechanisms in chromatin.