ROLE OF ISOLATED NUCLEAR PROTEINS IN DNA REPAIR

Project Details

Description

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.
StatusFinished
Effective start/end date9/30/868/31/91

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)

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