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 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

DLD

1+2

HCT116

+3

DLD

1+2MMR - Cell Lines

HCT116

DLD

1

DLD

1

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

DLD

1+2

HCT116

+3

DLD

1+2

MMR - Cell Lines

HCT116

DLD

1

DLD

1

HCT116

250

150

100

75

50

37

25

MW (kDa)

DMSO BPDE BPDEDMSO

PChk1

Results

Possible PChk1 signal

250

150

100

75

50

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