MOLECULAR ANALYSIS OF DROSOPHILA AP ENDONUCLEASES

Project: Research project

Description

Reactive oxygen species produced by ionizing radiation introduce a variety
of lesions in DNA including strand breaks, abasic (AP) sites, and
fragmented deoxyribose forms including 3-phosphoglycolate esters and 3'-
phosphomonoesters. These modified 3'-termini block DNA synthesis if not
identified by a class or direct acting enzymes called AP endonucleases
that are capable of removing blocked 3'-termini, as well as initiating the
repair of unmodified AP sites that arise spontaneously or as the product
of DNA glycosylases. Since AP sites are potentially mutagenic, AP
endonucleases are important enzymes to restore genetic integrity. The aims
outlined in this proposal are intended to broaden our knowledge or abasic
DNA repair in the genetically well-established eukaryotic organism
Drosophila melanogaster. Three AP endonucleases in Drosophila will be
studied, namely AP3, AP endonuclease I, and AP endonuclease fl. The cDNA
encoding AP3 has already been cloned. AP3 is bound to both nuclear
matrices as well as ribosomes. Its human homologue is ribosomal
phosphoprotein PO, which is elevated in patients suffering from the
autoimmune disease lupus. PO is also elevated in certain DNA-repair
deficient human tumor cell lines, thus drawing a link between certain
autoimmune diseases and DNA repair. The spectra of DNA damage recognized
by this AP endonuclease will be evaluated using a number of different
modified DNA substrates including lesions produced by ionizing radiation.
The other two AP endonucleases will be cloned using either an antibody
that specifically cross-reacts with these proteins, or oligonucleotides
generated to specific regions of the purified proteins. Once cloned,
products will be tested for biochemical activity similar to tests used for
defining the lesions identified by AP3. All three cDNA's will be used to
identify the genomic organization and upstream regulatory regions
associated with these enzymes and allow us to examine how these genes
respond to various environmental stimuli. Genetically, we will establish
the chromosomal location of these genes through in situ hybridization, and
from the knowledge gained from these studies target our search for mutants
deficient for each of the AP endonucleases in Drosophila. Mutants
identified will be subjected to various environmental agents to determine
which of these proteins dominates for a particular repair pathway.
Likewise, the rescue of E.coli mutants deficient for each of the AP
endonucleases will provide important information on the major role each of
these AP endonucleases play in the repair of DNA damaged by agents
generating reactive oxygen species along with alkylation damage.
StatusFinished
Effective start/end date9/30/939/29/98

Funding

  • National Institutes of Health
  • National Institutes of Health: $158,745.00
  • National Institutes of Health: $168,739.00
  • National Institutes of Health

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DNA-(Apurinic or Apyrimidinic Site) Lyase
Drosophila
DNA Repair
Ionizing Radiation
DNA
Reactive Oxygen Species
Enzymes
DNA Glycosylases
Deoxyribose
Proteins
Deoxyribonuclease I
Alkylation
Complementary DNA
Tumor Cell Line
Ribosomes
DNA Damage
In Situ Hybridization
Esters
Autoimmune Diseases
Escherichia coli

ASJC

  • Medicine(all)