investigating a role for dna mismatch repair in signaling a pah-induced dna replication arrest
DESCRIPTION
Investigating a Role for DNA Mismatch Repair in Signaling a PAH-Induced DNA Replication Arrest. Jacki L. Coburn Mentor: Dr. Andrew B. Buermeyer. Cancer affects us all. Lifetime risk for men: 1 in 2. Lifetime risk for women: 1 in 3. Excess risk factors: - PowerPoint PPT PresentationTRANSCRIPT
Investigating a Role for DNA Mismatch Repair in Signaling a PAH-Induced DNA Replication
Arrest
Jacki L. CoburnMentor: Dr. Andrew B. Buermeyer
Cancer affects us all
Lifetime risk for women: 1 in 3Lifetime risk for men: 1 in 2
Excess risk factors:• Mismatch repair deficiency (Lynch Syndrome)• Polycyclic aromatic hydrocarbon (PAH)
exposure
Mismatch Repair
• 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)
PAHs – they’re everywhere
Benzo[a]pyrene (B[a]P)
• 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-7,8-dihyrodiol-9,10- epoxide
Benzo[a]pyrene
CYP1A1
Epoxide Hydrolase
BPDE bonds to DNA and forms a bulky adduct
B[a]P-Adducted GuanineBPDE Lesion on DNA
Image courtesy of Peter HoffmanImage courtesy of Zephyris
A
Consequences of BaP-Derived Adducts
Pol δ
PCN
A G
NH
CA
CG
T
T
PCN
A
Pol κ
S-Phase Checkpoint Signaling
ATR ATR
Chk1
Chk1
P
Apoptosis
DNA Repair
Inhibition of Firing at Origins of Replication
DNA AdductsStalled
Replication Forks
P
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)
Predictions
• 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)
DLD1 – 2 defective copies of MSH6 (Chr. 2)
HCT116+3 – 2 defective copies of MLH1 (Chromosome 3) + 1 copy of WT MLH1 + neomycin resistance
gene
DLD1+2 – 2 defective copies of MSH6 (Chromosome 2) + 1 copy of WT MSH6 + neomycin resistance
gene
WT MLH1 Chr. 3 + neomycin resistance
gene
WT MSH6 Chr. 2 + neomycin resistance
gene
Experimental procedure
HCT116+3
MMR + Cell Lines
DLD1+2
HCT116+3
DLD1+2MMR - Cell Lines
HCT116
DLD1
DLD1
HCT116
250
150
100
75
50
37
25
MW (kDa)
Cultured cells:HCT 116 HCT116+3DLD1DLD1+2
BPDE (test)
DMSO (control)
Whole cell lysates
Gel electrophoresis and transfer to PVDF
membrane
Chemical treatment
Protein immunoblot to detect PChk1
DMSO BPDE BPDEDMSO
Assessing S-phase checkpoint activation: anticipated results
HCT116+3
MMR + Cell Lines
DLD1+2
HCT116+3
DLD1+2
MMR - Cell Lines
HCT116
DLD1
DLD1
HCT116
250
150
100
75
50
37
25
MW (kDa)
DMSO BPDE BPDEDMSO
PChk1
Results
Possible PChk1 signal
250
150
100
7550
37
25
MW (kDa)
Immuno-blot probed with anti-PChk1 (S345) polyclonal antibody
• MMR proficient and deficient cells show similar activation of S-phase checkpoint (dose dependent increase in PChk1 signal)
• Surprisingly, MMR-deficient cells show prolonged accumulation of PChk1, suggesting prolonged activation of checkpoint signaling
GAPDH
+/200/48
-/200/48
+/200/24
-/200/24
+/100/48
-/100/48
+/100/24
-/100/24
+/0/48
-/0/48
+/0/24
-/0/24 -/100/24Exposure time
[BPDE] (nM)
MM
R status
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 cellsImmunodepleted cell lysatesPurified Chk1
Future Research
• Investigate other markers of S-phase checkpoint activation and duration
• Analyzing downstream effects of prolonged checkpoint activation
Acknowledgements
• 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