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 3 Lifetime 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 Guanine BPDE Lesion on DNA Image courtesy of Peter Hoffman Image courtesy of Zephyris

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

S-Phase Checkpoint Signaling AT R Chk 1 P P Apoptosis DNA Repair Inhibition of Firing at Origins of Replication DNA Adducts Stalled Replication Forks P 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 HCT MMR + Cell Lines DLD1+2HCT DLD1+2 MMR - Cell Lines HCT116DLD1 HCT MW (kDa) Cultured cells: HCT 116 HCT116+3 DLD1 DLD1+2 BPDE (test) DMSO (control) Whole cell lysates Gel electrophoresis and transfer to PVDF membrane Chemical treatment Protein immunoblot to detect PChk1 DMSO BPDE DMSO

Assessing S-phase checkpoint activation: anticipated results HCT MMR + Cell Lines DLD1+2HCT DLD1+2 MMR - Cell Lines HCT116 DLD1 HCT MW (kDa) DMSO BPDE DMSO PChk1

Results Possible PChk1 signal 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 -/100/24 Exposure time [BPDE] (nM) MMR 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 cells Immunodepleted cell lysates Purified 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