Project Details


The goal of this proposal is to elucidate the molecular mechanisms in
human cells responsible for repair of a specific type of lesion
introduced into DNA by ultraviolet (UV) radiation, the pyrimidine
dimer. Of the various DNA lesions produced by UV radiation in sunlight,
the pyrimidine dimer is especially important in the etiology of sun-
induced skin cancer as well as a number of other pathological effects.
It is also among the DNA adducts most increased by depletion of the
atmospheric ozone layer. We have isolated a DNA endonuclease complex
from normal human cell chromatin which is composed of both a DNA
endonuclease with specificity for pyrimidine dimers and a protein
necessary for interaction of this endonuclease with damaged nucleosomal
DNA. This complex scans the DNA molecule so as to identify damaged sites
in a processive manner. We have shown that, in cells derived from
patients with the sun-sensitive, cancer prone, DNA repair-deficient
genetic disease, xeroderma pigmentosum, complementation group A (XPA),
this endonuclease complex both is deficient in ability to incise damaged
nucleosomal DNA and is unable to scan the damaged DNA molecule, instead
recognizing damaged sites in a distributive manner. The proposed studies
will investigate the molecular mechanisms by which the proteins in this
complex interact with each other and with damaged nucleosomal DNA to
initiate the repair of pyrimidine dimers in normal human cells. XPA
cells, and their defective endonuclease complex, will be used as a model
system in which these interactions are deficient. The proteins within
the normal DNA endonuclease complex will be identified and isolated.
cDNAs coding for each of these proteins will be cloned, sequenced and
expressed in cultured human cells to confirm their role in DNA repair
processes. The chromosomal localization of the genes for each of these
proteins will be determined by in situ hybridization. The presence of
mutations in the cDNAs coding for these proteins in XPA cells will be
analyzed. Both the normal and XPA proteins will be purified by
overexpressing the cDNAs of each in E. coli. Specificity of binding of
each of these proteins, separately and then in combination, to site
directed pyrimidine dimers on nucleosomal and non-nucleosomal DNA will
be compared. The influence of the normal and XPA chromatin-interacting
protein on the mode of interaction of each endonuclease with damaged
naked and nucleosomal DNA will be examined. This unique combination of
approaches will allow us to obtain valuable insight into the mechanisms
responsible for repair of UV radiation damage in DNA.
Effective start/end date9/30/929/29/98


  • National Institutes of Health
  • National Institutes of Health: $245,617.00
  • National Institutes of Health: $235,563.00
  • National Institutes of Health
  • National Institutes of Health


  • Environmental Science(all)
  • Medicine(all)

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