Investigating a Role for DNA
Mismatch Repair in Signaling a
PAH-Induced DNA Replication
Arrest
Jacki L. Coburn
Mentor: Dr. Andrew B. Buermeyer

Lifetime risk for men: 1 in 2 Lifetime risk for women: 1 in 3
Excess risk factors:
•
Mismatch repair deficiency (Lynch Syndrome)
•
Polycyclic aromatic hydrocarbon (PAH) exposure

• Highly conserved pathway primarily focused on the repair of replication errors
• Conserved MMR specific constituent proteins include Mut Sα (MSH2-MSH6) and Mut Lα
(MLH1-PMS2)
• MMR deficiency has significant impacts on human health (Lynch Syndrome)

•
Best known and most studied of PAHs
•
Volatilized during combustion of organic compounds
•
Detected in air, water, food and soil
•
Highly mutagenic and carcinogenic

B[a]P is converted to a diol epoxide (BPDE) through enzymatic action
Benzo[a]pyrene
CYP1A1
Epoxide
Hydrolase
(+)-benzo[a]pyrene-7,8dihyrodiol-9,10- epoxide

BPDE bonds to DNA and forms a bulky adduct
BPDE Lesion on DNA
Image courtesy of Zephyris
B[a]P-Adducted Guanine
Image courtesy of Peter Hoffman

Consequences of BaP-Derived Adducts
A
T
C
Pol δ
C
NH
G
T
A
Pol κ

DNA Adducts
Stalled
Replication
Forks
AT
R
AT
R
P
Chk
1
Chk
1
P
Apoptosis
Inhibition of
Firing at Origins of Replication
DNA Repair

Hypothesis:
MMR participates in signaling S-phase checkpoint in response to BPDE exposure.
(MMR may participate in recruitment of ATR)
Alternate Hypothesis:
MMR helps turn off S-phase checkpoint.
(MMR may promote resolution of stalled replication forks)

• MMR deficient cells will show less activation of S-phase checkpoint in response to BPDE exposure.
– MMR deficient cells will display lower levels of
PChk1.
– PChk1 can be measured using semi-quantitative immuno-blotting.

Model System: MMR deficient and proficient cell lines
HCT116 – 2 defective copies of MLH1 (Chr. 3)
WT MLH1 Chr. 3 + neomycin resistance gene
HCT116+3 – 2 defective copies of
MLH1 (Chromosome 3) + 1 copy of
WT MLH1 + neomycin resistance gene
DLD1 – 2 defective copies of MSH6 (Chr. 2)
WT MSH6 Chr. 2 + neomycin resistance gene
DLD1+2 – 2 defective copies of
MSH6 (Chromosome 2) + 1 copy of
WT MSH6 + neomycin resistance gene

MW
(kDa)
250
37
25
75
50
150
100
Cultured cells:
HCT 116
HCT116+3
DLD1
DLD1+2
BPDE (test)
DMSO (control)
Chemical treatment Whole cell lysates
MMR - Cell Lines MMR + Cell Lines
DMSO BPDE DMSO BPDE
Protein immunoblot to detect PChk1
Gel electrophoresis and transfer to PVDF membrane

Assessing S-phase checkpoint activation: anticipated results
MW
(kDa)
250
37
25
75
50
150
100
MMR - Cell Lines
DMSO BPDE
MMR + Cell Lines
DMSO BPDE
PChk1

Immuno-blot probed with anti-PChk1 (S345) polyclonal antibody
MW (kDa) -/100/24
75
50
37
25
250
150
100
Possible PChk1 signal
GAPDH
•
MMR proficient and deficient cells show similar activation of Sphase checkpoint (dose dependent increase in PChk1 signal)
•
Surprisingly, MMR-deficient cells show prolonged accumulation of PChk1, suggesting prolonged activation of checkpoint signaling

Confirming the identity of the signal as
PChk1
Positive controls:
HeLa cells treated with UV radiation
HeLa cells treated with etoposide
Negative controls:
Chk1 knockdown cells
Immunodepleted cell lysates
Purified Chk1

•
Investigate other markers of S-phase checkpoint activation and duration
•
Analyzing downstream effects of prolonged checkpoint activation

•
Dr. Kevin Ahern
•
Dr. Andrew B. Buermeyer
•
Frances Cripp Scholarship Fund
•
Peter Hoffman
•
Casey Kernan
•
Fatimah Almousawi
•
Kimberly Sarver
•
HHMI
•
URISC
•
Dr. Anthony C. Zable