Impact of PAH Exposure on
Accumulation of Translesion
DNA Polymerases in Mouse
Colon
Jacki L. Coburn
Mentor: Dr. Andrew B. Buermeyer
Colorectal Cancer
• In the U.S., 70,270 men and 68,857
women were diagnosed with colorectal
cancer in 2006.
• The same year, 26,801 men and
26,395 women died of colorectal
cancer in the U.S.
• Exposure to polycyclic aromatic
hydrocarbons (PAHs) is associated
with increased risk of developing
colon cancer.
Benzo[a]pyrene (B[a]P)
• Volatilized during combustion of organic
compounds
• Present in coal tar and diesel exhaust
• Highly mutagenic and carcinogenic
Routes of Exposure
Inhalation (air pollution, use of
tobacco products)
Dermal contact (soot and coal tar
derivative products)
Ingestion (contaminated water,
cooked meat products and
vegetable oils)
Levels of B[a]P Found in
Selected Foods*
Sample
PPB (ng/g)
BBQ Steak, Very Well Done
4.75
BBQ Chicken (bone and skin), Well Done
4.57
BBQ Hamburger, Medium
0.56
Pumpkin Pie
0.47
Fast Food French Fry
0.22
Tomato (Fresh)
0.19
*Kazerouni N., Sinha R., Hsu C.-H., Greenberg A., Rothman N.Analysis of 200 food items for benzo[a]pyrene and estimation of its intake in an epidemiologic study(2001)ﾊ
Food and Chemical Toxicology,ﾊ39ﾊ(5),ﾊpp.ﾊ423-436.
B[a]P is converted to its diol epoxide (BPDE)
through cellular enzymatic action.
Cytochrome
P4501A1
Epoxide
Hydrolase
Benzo[a]pyrene
(+)-benzo[a]pyrene7,8-dihyrodiol-9,10epoxide
BPDE bonds with DNA to form
a bulky adduct
BPDE Lesion on DNA
Image courtesy of Zephyris
B[a]P-Adducted Guanine
Image courtesy of Peter Hoffman
Impact of B[a]P Adduct on DNA
Synthesis
• Replication stalls at B[a]P adduct site
• Translesion polymerases (TLPs) are
recruited for synthesis
• Polymerase  (Pol) - B[a]P adducts
• Polymerase  (Pol) - UV photodamage
The ProblemWith TLPs
• Specialized TLPs preferentially add correct bases
at specific lesion sites, but replicate in an errorprone manner downstream from the lesion. Why?
• Larger active site
• Lack of proof-reading ability
Pol almost exclusively inserts C opposite B[a]P-adducted
G, while Pol inserts bases at random, and Polι
preferentially inserts G.
?
Rechkoblit,O et al. J. Biol. Chem. 2002;277:30488-30494
©2002 by American Society for Biochemistry and Molecular Biology
Regulation of TLPs
• Pol is known to be stabilized by UV radiation
and ubiquitin-tagged for destruction by
proteasomes
• Regulation of Pol is not as well understood
• How specific TLPs are recruited for bypass of
specific lesions is unknown
• Local concentration of TLPs may be important in
selection process
Hypothesis
Pol levels are higher in BPDE-induced
target cells because Pol can accurately
bypass bulky adduct lesions such as those
formed by PAHs.
Predictions
• Pol levels will be greater in BPDE-treated target
cells than in negative control target cells.
• Levels of Pol will increase relative to Pol levels
when induced by BPDE.
• Levels of Pol will be very low, if not
undetectable.
• Pol levels will peak shortly after dosing, then
slowly taper off
Research Questions
• What is the normal, uninduced level of Polη and
Pol in the target cells?
• How do Polη and Pol levels in the target cells
change with increasing dosages of BPDE?
• How many hours after dosing does the Pol level
peak?
Experimental Design
Test subjects are Pol/Polη proficient wild-type laboratory mice, with one
negative control group and test groups given different doses of BPDE.
Negative
Control
(No BPDE)
Mice are harvested at different times
following exposure to BPDE
0 hr
8 hr
2 hr
4 hr
12 hr
24 hr
Quantitative Western Blots
Pol
Lysate
Pol
Lysate
Pol
Lysate
Lysate
Lysate
Pol K/O
Cell
Pol
Pol
Pol
Pol
Lysate
Pol
Lysate
Pol
Lysate
Lysate
Lysate
Pol K/O
Cell
Pol
Pol
Chemiluminescence
Pol
Chemiluminescence
Standard Curves
Interpolating Polymerase Levels in
Mouse Extracts
Negative
Control
BPDE
BPDE
BPDE
(No BPDE
Pol
Chemiluminescence
Mouse Colon Extracts
Pure Pol
Mouse Colon
Pol
Negative
Control
(No BPDE
Pol
BPDE
BPDE
BPDE
Chemiluminescence
Mouse Colon Extracts
Pure Pol
Mouse Colon
Pol
Expected Relationships
Temporal Variation of Polymerase
Levels after BPDE Dosing
Predicted BPDE-Induced
Accumulation of Pol
Pol
Pol
Pol
Undetectable
Undetectable
Polymerase Level
Polymerase Level
Pol
Time
Increasing Dose of BPDE
Detection of Pol
250
250
250
150
150
150
150
100
100
100
75
75
75
50
50
50
Ladder
6.25 g
HeLa
12.5 g
HeLa
25 g
HeLa
50 g
HeLa
Signals at 90 kDa detected by a mouse
monoclonal Pol antibody.
Ladder
40 ng
80 ng
Pol
Pol


Ladder
Full-length purified protein
does not co-migrate with signal
in HeLa (100 kDa)
Detection of Polη
250
250
250
250
150
150
150
150
100
100
100
100
75
75
75
75
50
50
Polη signal is very weak in HeLa and
may not be detectable in mouse tissues
50
50
Non-specific signal from
secondary antibody comigrates with Polη
Benefits of this research
• First quantification of Pol and Polη in normal and
B[a]P induced cells
• Understanding of dose-response relationship and
temporal variance of Pol BPDE in target cells
• Will inform research on the mechanism of TLP
recruitment.
• Will inform research on normal cellular responses
triggered by exposure to PAHs such as B[a]P
What’s Next
• Quantify full-length protein fraction of purified
Pol and Polη
• Test rabbit Pol and Polη antibodies to avoid
nonspecific interference from secondary
antibodies in the mouse extracts.
• Create standard curves
• Dose mice and analyze extracts
Acknowledgements
•
•
•
•
•
•
•
•
Dr. Andrew B. Buermeyer
Dr. John Hays and the Hays Lab
HHMI
Cripps Scholarship Fund
Joshua T. Robinson
Dr. Vidya Schalk
URISC
Dr. Anthony C. Zable
References
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