BIOCHEMISTRY AND BIOLOGY OF AN XP-A RELATED GENE PRODUCT

A major pathway by which human cells repair bulky DNA damage is the
nucleotide excision repair pathway. The genetic and biochemical complexity
of this process In humans has been revealed through the study of
individuals with the cancer-prone genetic disease, xeroderma pigmentosum
(XP). In XP cells, excision repair is partially to completely compromised,
depending on which of at least 7 genes are affected. Recently two
different genes have been cloned which, when individually introduced into
XP-A cells, give rise to substantially enhanced levels of survival after UV
irradiation challenge. One of these genes, that was identified in my
laboratory, has been shown to restore the ability of XP-A cells to repair a
variety of bulky lesions: cyclobutane dimers, anti-BPDE adducts and
N-AcO-AAF lesions. However, In these same cells, restoration of (6-4)
photoproduct repair proceeds at approximately 3% the rate that is measured
in normal human cells. The objective of this research is to provide a basic understanding of the
genetics and biochemistry of the mechanism(s) by which normal human cells
repair bulky DNA modifications. Although our preliminary studies focused
on observations concerning the phenotype of partially repair-proficient
XP-A cells, a major component of this proposal focuses on the
XP-A-correcting gene and its gene product. The complete XP-A-correcting
gene will be identified from lamda/gt11 cDNA libraries, using the partial
cDNA sequence that is already available. This complete cDNA wall be
tailored for mammalian cell expression and reintroduced Into XP-A cells and
those cells evaluated for enhanced resistance to a variety of DNA-damaging
agents. With the gene sequence available, antibody reagents will be
produced that will facilitate determining the role that this gene product
plays in the initiation of repair and its interaction with other cellular
proteins. Antibodies will also facilitate the purification of this protein
either from human tissues or from a bacterial expression system. The
purified protein will be used in vitro reconstruction assays and in
biophysical characterization. In addition to these genetic and biochemical studies, a further
understanding of this gene is warranted through cell biology studies.
Specifically, new cell lines will be created in which both XP-A-
complementing genes are introduced into the same cell-these cells should
display full DNA-repair responsiveness. Also, preferential DNA repair
studies will be performed that are designed to probe whether the overall
repair process or some subset of repair (i.e., active gene ret)air) has
been restored.