• Zarbl, Helmut (PI)
  • Essigmann, John (PI)
  • Fitts, Renee (PI)
  • Zarbl, Helmut (PI)
  • Thilly, William (PI)
  • Thilly, William (PI)
  • Demple, Bruce (PI)
  • Samson, Leona (PI)
  • Thilly, William (PI)
  • Walker, Graham (PI)
  • Loechler, Edward (PI)
  • Demple, Bruce (PI)
  • Essigmann, John (PI)
  • Samson, Leona (PI)
  • Walker, Graham (PI)
  • Demple, Bruce (PI)
  • Samson, Leona (PI)
  • Thilly, William (PI)
  • Walker, Graham (PI)
  • Loechler, Edward (PI)
  • Demple, Bruce (PI)
  • Loechler, Edward (PI)

Project Details


Spontaneous mutations contribute significantly to human genetic disease
and form the background against which the genetic toxicity of
environmental agents must be judged. In spite of these central concerns,
the cellular processes that influence the spontaneous mutation rate are
poorly understood. This project addresses key issues regarding the
biochemical mechanisms of spontaneous mutagenesis eukaryotes: Is
spontaneous mutation affected by processing of a gene by transcription and
replication (Aim 1)? Is spontaneous mutation driven by metabolically
generated DNA damage such as oxygen radicals (Aims 2-4)? Do repairable
spontaneous DNA damages escape correction and cause mutations in normal
cells (Aim 3)? Is their probability of generating mutations more a
question of kinetics (repair vs. replication) or the rate at which damage
is generated (Aim 4)? Do mutations due to endogenous DNA damage result
from active processing pathways that handle environmental mutagens (Aim
5)? To achieve these ends, we will exploit our ability to modulate the
DNA repair capacity of the yeast Saccharomyces cerevisiae by inactivating
the APN1 gene we cloned previously, which encodes the main endonuclease
for abasic sites in this organism. Strains lacking this activity have a
substantially elevated rate of spontaneous mutation, which is generated in
part by abasic sites produced by a DNA glycosylase (the MAG gene product)
acting on endogenous DNA damages. We will construct test strains bearing
a mutation targets of human origin, in common with the other projects.
Rapid determination of mutation spectra using a newly-developed approach
will allow us to tease out specific events that limit normal genetic
stability. These targets will be inserted with different orientations
respective to replication and transcription (leading vs. lagging strand,
and transcribed vs. nontranscribed strand). The role of endogenous damage
will be assessed by deleting the APN1 or MAG genes, and by culturing the
cells under different conditions (aerobic vs. anaerobic, etc.). Yeast
strains will also be constructed with increased levels of Apn1 protein or
the human abasic endonuclease, Ape, which will reveal whether repair of
such damages is limiting in normal cells. Finally, mutations in the RAD6
or REV3 genes will be introduced to reveal whether active mutational
systems contribute to "unprogrammed" genetic change.
Effective start/end date1/1/018/31/99


  • Environmental Science(all)
  • Medicine(all)
  • Genetics
  • Molecular Biology
  • Biochemistry
  • Spectroscopy
  • Oncology
  • Cancer Research
  • Toxicology