B.S. Northwestern University, 2005
Year Graduate Student
Advisor: Bruce Demple
Graduate Program: Genetics
Title: The contribution of various DNA damages to
DNA within our cells can be damaged in numerous ways. Chemical damages
include alkylation, oxidation, and hydrolysis, all of which can originate
from normal metabolism. Exposure to UV radiation or environmental toxins
can also cause DNA damage. At a cellular level, such damages can lead
to cell cycle arrest, apoptosis, or mutations that modify gene expression.
Repair mechanisms such as base excision repair (BER) have evolved to
reduce the effects of DNA damage on cell survival. BER is the major
repair pathway for small single-base lesions. At least 10,000 such lesions
are repaired through BER in each human cell per day. To initiate BER,
a DNA glycosylase detects and removes a damaged nucleobase, leaving
behind an apurinic/apyrimidinic (AP) site. AP endonuclease (APE1) nicks
the sugar-phosphate backbone at the 5’ end of the AP site. DNA
polymerase ß replaces the proper missing nucleotide. Finally,
DNA ligase completes the repair by joining the sugar-phosphate backbone
with a new phosphodiester bond.
with single genetic knockouts of the DNA glycosylases exhibit little
or no phenotypic change compared to wild-type animals. Uracil glycosylase
(UNG) deficient mice have a 100-fold increase in uracil misincorporation
during cell replication, yet are healthy for the first 12 months of
life. 8-oxoG glycosylase (OGG1) deficient mice have a three-fold increase
in 8-oxoG. However, the accumulation of 8-oxoG is not associated with
tumorigenesis (Barnes & Lindahl, 2004). In contrast with glycosylase
knockouts, genetic knockout of APE1, DNA polymerase ß, or DNA
ligase 1 in mice is embryonic lethal (Xanthoudakis, Smeyne, Wallace,
& Curran, 1996) (Sugo, Aratani, Nagashima, Kubota, & Koyama,
2000)(Bentley et al., 1996). The vital role of Ape1 depends on its DNA
repair activity and is observed even at the cellular level; severe APE1
knockdown reduces cell proliferation and promotes apoptosis (Fung &
Demple, 2005). It appears that the presence of unrepaired AP sites is
more toxic to the cell than are damaged nucleobases. We do not know,
however, what types of damage have greater toxic effects to APE1-deficient
cells. One of the goals is to understand the contributions that different
damaged bases have towards cell toxicity.
We are using
several approaches to study the contribution that various types of DNA
damage have on cell toxicity. One method we are using to determine the
contribution of oxidative damage on DNA is incubating U2OS and HeLa
cell lines under 21% O2 and 1% O2. Cells that have been grown under
hypoxic conditions should have lower levels of reactive oxygen species
and decreased numbers of nucleobases damaged by ROS. In a preliminary
experiment using quantitative PCR, we have shown that cells grown under
1% O2 have between a 2- to 4-fold decrease in the level of the mRNA
transcripts of the UNG2 (uracil-DNA glycosylase) and APE1 genes compared
to cells grown under 21% O2. I am replicating this experiment, and will
check this observation with western blotting to assess the Ung2 and
Ape1 protein levels. I will continue to explore whether changes in O2
affect the proliferation rate of Ape1-deficient cells. Future studies
include knocking down DNA glycosylases such as UNG2 and OGG1 and quantifying
the contribution of glycosylases to cell proliferation in Ape1-deficient
(pre-MSTP publications indicated with an *)
CC , Chen KY , Breslow JL. (2008). Activation of pregnane
X receptor induces hypercholesterolemia and accelerates atherosclerosis
in mice. ATVB. Jun; 28(6):E32
S , Teupser D , Tan M , Chen KY , Breslow JL. (2007).
The protective effect of A20 on atherosclerosis in apolipoprotein E-deficient
mice is associated with reduced expression of NF-kappa B target genes.
PNAS. Nov. 20; 104(47):18601-18606.
SL, Chen KY, Chenn A. (2005). A cell behavior screen:
identification, sorting, and enrichment of cells based on motility.
BMC Cell Biol. 6:14.