1. Kinetics of MutSα-Mediated DNA Mismatch Repair William Gibson Dr. John Hays Oregon State University Summer 2010

2. The Importance of DNA Mismatch Repair (MMR) A poorly working MMR system can cause many problems such as accelerated cell aging, cell death, and cancer. Diseases caused by defects in MMR systems are costly and damaging because of their severity and the lack of treatment options available.

3. MutSα-MMR System MutSα-MMR system 3 main components MutSα – Two subunits which form a dimer to bind DNA mismatches or adducts. MutLα – Two subunits which assist MutSα and exhibit a nuclease function. PCNA – A trimeric clamp which helps guide MutSα and MutLα along the DNA strand. Mut-Lα Mut-Sα PCNA

4. DNA Damage from PAH Alkylation Polycyclic aromatic hydrocarbons (PAHs) attach to DNA and cause lesions. PAH's are produced by the combustion of complex carbon compounds (ex. coal).

5. What I Want to Do Examine MMR initiation at gaps vs. nicks. Examine how the MMR system responds to adducts such as a PAH-guanine (PAH-G) adduct. Determine if PAH-G mismatches provoke MMR excision similar to standard base mismatches.

6. Plasmid Model for MMR Initiation: MutSα complexes with MutLα and PCNA. Proximal Excision: MutSα-MMR complex excises to the mismatch. Distal Excision: MutSα-MMR complex excises past mismatch before dissociating.

7. Plasmid Construct The plasmid construct has multiple nicking and gapping sites to initiate repair. PAH-Guanine is opposite to any of the 4 bases (A, G, T, C)

8. Experimental Design Growth of Plasmid ↓ Addition of Gap and Mismatch Sites ↓ MMR Assay (Using Radio-Labeled Reporter Oligos) ↓ Results Analysis (Compare Gapped vs. Nicked and 3 Different Mismatches vs. Controls)

9. Growth of Plasmid Addition of Gap and Mismatch Sites MMR Assays Results Analysis Growth of Plasmid The plasmid is modified from the highly used pUC19 plasmid. The plasmid contains a drug resistance gene.

10. Growth of Plasmid Addition of Gap and Mismatch Sites MMR Assays Results Analysis Gapping and Nicking Multiple unique restriction sites allow different areas to be targeted. Modified restriction enzymes allow nicking of a single strand at nick sites.

11. Growth of Plasmid Addition of Gap and Mismatch Sites MMR Assays Results Analysis MMR Assay 1 Excision Assay: Measure Gapped vs. Nicked initiation. RepairNo Repair

12. Growth of Plasmid Addition of Gap and Mismatch Sites MMR Assays Results Analysis MMR Assay 1 No RepairRepair Time 0Time 2Time 4Time 6

13. Growth of Plasmid Addition of Gap and Mismatch Sites MMR Assays Results Analysis MMR Assay 2 PAH Assay: Measure relative abundance of the radiolabeled probes. Excision past PAH Excision but not past PAH No excision triggered

14. Growth of Plasmid Addition of Gap and Mismatch Sites MMR Assays Results Analysis Results Still Pending The first assay will provide a definitive answer on gap vs. nick initiation, which will allow future assays to use the most efficient initiation method. By exploring the behavior of the MMR system in regards to PAH-G adducts, specific kinetic models can be built to further understand the MMR process at a molecular level.

15. Acknowledgments Dr. John Hays and Peter Hoffman Colin Tominey Dr. Mark Curtis, Johanna Steinbrecher, Robert Ursu, Ana Brar, Taylor Hoffman, Buck Wilcox Figures: ATSDR (slide 2), JBC (slide 4), myself (slide 3,6,7,11,12,13), NEBcutter (slide 10), David Brooks (slide 9) Special thanks to Kevin Ahern and the HHMI program for letting me take part!