1. Biochemical Defects Associated with Cancer-Causing Pathogenic Mutations in Human MLH1 Andrew Nguyen Laboratory of Dr. Andrew Buermeyer Department of Environmental and Molecular Toxicology Oregon State University

2. ©2005 American Cancer Society, National Cancer Institute Deaths Caused by Cancer – United States, 2005 Approximately 56,000 deaths from colorectal cancer in 2005 alone Women 273,560 deaths Colon and rectum10% Men 291,270 deaths

3. Why DNA repair is important DNA can be damaged by mutagens (ex: UV light, Radiation, Chemicals) DNA replication is error prone – base pair mismatches are sometimes introduced by DNA Polymerase Lack of correction can lead to mutations, a risk factor for cancer

4. Consequences of DNA Mismatch Repair Deficiency Accumulation of mutations in DNA can cause cancer. Loss of mismatch repair (MMR) is linked to significantly increased cancer risk in a condition known as Lynch Syndrome. This disorder is characterized by a predisposition to early onset of colorectal and other internal cancers. Knowing who is at risk for cancer because of MMR deficiency can influence treatment options for existing patients or surveillance strategies for higher risk people.

5. DNA Mismatch Repair (MMR) MMR acts to maintain genomic integrity by correcting DNA replication errors Evolutionarily conserved process Mechanism involves several protein dimers necessary for successful repair MutLα is composed of two separate proteins, MLH1 and PMS2

6. Mechanism of Mismatch Repair Recognition Excision Resynthesis G T T T A MutS MutL Exonuclease DNA polymerase In-Vitro strand choice is determined by nick in non-template strand

7. Project Goals Goals: Establish experimental conditions for an in-vitro repair assay Compare four MLH1 mutants identified in human cancers vs. wild type MLH1 to see how/if the mutations affect MMR These mutants are: K751R, R755W, L582V, L607H [original amino acid] [location of mutation] [new amino acid] Approach: In-vitro repair assay with MP1 extract (MutLα deficient) plus recombinant MutLα Questions: What level of DNA repair activity will the mutants exhibit compared to the wild type? Amount of substrate repaired or kinetics of repair, for example

8. Experimental Approach A plasmid containing a single known mismatch will be the substrate for the repair assays Recognition and correction of mismatch will restore the Xho1 restriction site on the plasmid MMR activity assessed by percentage of plasmid substrate that can be restricted ResynthesisRestriction Ban1 restriction site Excision % repair 050100 Ban1 restriction site

9. Creation of Mismatch Containing Plasmid Substrate pRO1 Start with the pRO1 plasmid Nick plasmid Generate gap by adding excess of complementary oligonucleotide Anneal a DNA fragment of known sequence that will create a mismatch Mismatch substrate

10. Experimental Approach – MP1 titration MP1 cellular extract contains all MMR factors that are required for repair except MutL α A titration was done to identify conditions under which these factors aren’t limiting for repair Increasing amounts of MP1 added 0 No MutL Repair saturated around 225-250 µgRepair is MutL, MP1 dependent Question: How much non-MutL containing cell extract is saturating?

11. Experimental Approach – MLH1 WT Kinetics After 25 minutes no additional repair was observed Identical repair reactions stopped at different times Minutes 0 5 10 15 20 25 30 35 40 45 50 55 60 Question: How does repair progress with time?

12. Results – MLH1 Mutant Endpoint Assay R755W appears to exhibit no repair at all L607H, L582V, K751R seem to repair about the same as WT 2200 bp 1150 bp 1050 bp L607HL582VK571RR755WWT No MutL Question: Given a saturating amount of time, what levels of repair will the mutants exhibit?

13. Results – MLH1 Mutant Endpoint Assay continued.. L607HL562VK751RR755WWT Negative repair results from subtraction of negative control (unidentified repair pathway)

14. Summary -MutLα is required for repair -225-250 µg MP1 extract is saturating -Repair saturates around 25 minutes -MutLα can be overexpressed in an active form -Activity can be reliably assayed -Kinetics are commensurate with previously published data -L607H, L582V, K751R appear to be capable of wild type levels of repair -R755W appears to exhibit no activity

15. Future Studies -Refining the assay and protein quantitation -Expand substrate (CT, AG, AC, 8-oxo G, T dimers) -Different mutants

16. Thanks to: Dr. Andrew Buermeyer Dr. Scott Nelson Dr. Kevin Ahern Howard Hughes Medical Institute (HHMI) Undergraduate Research, Innovation, Scholarship & Creativity (URISC)