How tightly packed
chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged
DNA-binding protein (UV-
DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the
nucleosome repeat landscape of
chromatin. Therefore, the
nucleosomes of human cells have been dissected by
micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-
DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A
ubiquitin ligase activity is necessary to retain the
xeroderma pigmentosum group C (XPC) partner at such internucleosomal repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-
DDB also adopts a
ubiquitin-independent function is evidenced by domain mapping and in situ
protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable β-hairpin insertion of XPC into substrate
DNA. We conclude that the evolutionary advent of UV-
DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic
chromatin.