GENETIC ANALYSIS OF NUCLEOTIDE EXCISION REPAIR

  • Thompson, Larry H (PI)
  • Weber, Christine (PI)

Project: Research project

Project Details

Description

Defects in the nucleotide excision repair pathway, one of several
DNA repair systems, are responsible for the series of cancer-prone
genetic disorders called xeroderma pigmentosum (XP). The genetic and
biochemical complexity of this repair process Is reflected in the
existence of multiple complementation groups. The basis for future
biochemical studies of the structure and function of the mammalian
nucleotide excision repair protein ERCC2 will be provided by this study
to elucidate the multiple functions of ERCC22 and identify the role of
ERCC2 in the preferential repair of UV-induced DNA adducts. The ERCC2 protein has distinct roles in DNA repair, recombination,
and cell viability. ERCC2 mutants with defects in either 'he replication
or essential functions will be created by site directed mutagenesis and
targeted recombination using CHO cells, made possible since ERCC2 is
fortuitously single copy in these cells. Defects in the replication
function should result in increased levels of recombination and mutation.
This study will provide direct evidence for these functions in mammalian
cells. The generation of mammalian hyper-recombination mutants will
provide a valuable tool for future studies into this important process
that plays a critical role in mutagenesis and carcinogenesis. Characterization of four UV-sensitive hamster ERCC2 mutants has
revealed heterogeneity in the level of removal of (6-4)photoproducts,
suggesting a role for ERCC2 in the preferential repair of damage in
actively transcribed sequences. The specific molecular defect in the
ERCC2 gene of these four mutants and the change in three partial
revertants will be identified using PCR and direct sequence
determination. In order to accomplish the preceding goals, the nucleotide sequence
of cDNA clones and genomic intron/exon junctions and flanking regions
will be determined. The wild-type hamster ERCC2 clones for these
determinations will be isolated using a previously isolated human cDNA
probe In order to study the biochemical properties of the mutant ERCC2
proteins, the hamster ERCC2 gene will be cloned into a yeast expression
vector and site directed mutagenesis will be used to introduce the same
defects as are in the mutants described above. Mutant proteins produced
from these clones will be used for biochemical and enzymatic
characterization in subsequent experiments. Relating the biochemical
activities of the proteins to the molecular defects and cellular
phenotypes will provide insights into the functional domains of ERCC2
that are necessary for its various roles in DNA metabolism. Understanding the multiple roles for ERCC2 will provide insight into
the processes of DNA repair and metabolism, vital cellular processes for
maintaining genome integrity. In addition to furthering our
understanding of fundamental aspects of DNA metabolism, this study will
provide the foundation for constructing an animal model for studying the
relationship of differences in DNA repair capacity to differential
susceptibilities to carcinogenesis.
StatusFinished
Effective start/end date5/20/913/31/04

Funding

  • National Institutes of Health: $451,972.00
  • National Institutes of Health: $436,501.00
  • National Institutes of Health
  • National Institutes of Health: $462,515.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)

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