Our objective is to elucidate the molecular mechanisms in human cells responsible for repair of lesions introduced into DNA by short wavelength ultraviolet (254 nm; UVC) light. This problem will be approached using two new, proven systems, which we have developed, to 1) purify, characterize, and determine the site(s) of action of a DNA endonuclease activity, pI 7.6, with increased activity on UVC damaged DNA, which we have already isolated and partially purified from human lymphoblastoid cell chromatin, and to 2) examine the role of chromatin structure in modifying this endonuclease activity, using a reconstituted nucleosomal substrate. Normal human lymphoblastoid cells will be used as well as those from patients with xeroderma pigmentosum (XP), a disorder with marked sensitivity to sun and UV light and a marked tendency to develop sun related skin changes, including numerous cancers, in light exposed areas. Cultured cells from XP patients show markedly increased sensititivy to UVC light, which, in cells from most XP patients is related to a defece in the first, endonuclease mediated, step of the nucleotide excision mechanism for repair of UVC light-induced lesions in DNA. In at least one of the most severely affected complementation groups, A, of XP (XPA), however, there is evidence that the defect is not in the ability of the UV endonuclease to incise DNA when it is in the form of chromatin. Thus, although XP is perhaps the best available model for the effects of sunlight on human skin and for carcinogenesis due to exposure to an environmental agent, the molecular mechanisms responsible for this defect have remained obscure. Our approach should allow us to elucidate these mechanisms. It has already enabled us to ascertain that the UVC endonuclease activity at pI 7.6 is, in fact, present in XPA cells but that there is a defect in this endonuclease itself or in a closely associated cofactor which is needed for endonuclease activity on UVC irradiated DNA when it is in the form of chromatin. The studies will more clearly elucidate the precise role of chromatin structure in this UV endonuclease activity. Our unique combination of approaches should obtain valuable insight into the molecular mechanisms responsible for repair of UVC light damage to DNA, and into the mechanisms responsible for UV light effects on human skin.
