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Investigating a Role for DNA Mismatch Repair in Signaling a PAH-Induced DNA Replication Arrest

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:

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Investigating a Role for DNA Mismatch Repair in Signaling a PAH-Induced DNA Replication Arrest

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

  2. Cancer affects us all 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

  3. 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)

  4. PAHs – they’re everywhere

  5. 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

  6. B[a]P is converted to a diolepoxide (BPDE) through enzymatic action CYP1A1 EpoxideHydrolase Benzo[a]pyrene (+)-benzo[a]pyrene-7,8-dihyrodiol-9,10- epoxide

  7. 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

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

  9. S-Phase Checkpoint Signaling Stalled Replication Forks DNA Adducts P ATR ATR P Chk1 Chk1 Inhibition of Firing at Origins of Replication Apoptosis DNA Repair

  10. 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)

  11. 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.

  12. 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 WT MSH6 Chr. 2 + neomycin resistance gene DLD1+2 – 2 defective copies of MSH6 (Chromosome 2) + 1 copy of WT MSH6 + neomycin resistance gene DLD1 – 2 defective copies of MSH6 (Chr. 2)

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

  14. Assessing S-phase checkpoint activation: anticipated results MW (kDa) MMR - Cell Lines MMR + Cell Lines 250 150 100 75 50 37 25 HCT116+3 HCT116+3 DLD1+2 DLD1+2 HCT116 DLD1 HCT116 DLD1 PChk1 DMSO DMSO BPDE BPDE

  15. Results +/200/48 -/200/48 +/200/24 -/200/24 +/100/48 -/100/48 +/100/24 -/100/24 +/0/48 -/0/48 +/0/24 -/0/24 Immuno-blot probed with anti-PChk1 (S345) polyclonal antibody MW (kDa) -/100/24 Exposure time [BPDE] (nM) MMR status 250 150 100 75 Possible PChk1 signal GAPDH 50 37 • 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 25

  16. 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

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

  18. 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

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