1. Effect of Cancer-Associated Mutations on MLH1 Interaction with Exonuclease Gautam Mankaney Mentor: Dr. Andrew Buermeyer Howard Hughes Medical Internship

2. CANCER Cancer Uncontrolled proliferation of cells 2nd leading cause of death (500 thousand/ yr) Causes Cellular mutations Inherited or Sporadic Colorectal Cancer 3rd most common cancer (10.7%) $8.4 billion in treatment costs 3-5% cases linked to Lynch Syndrome (HNPCC) New Cancer Incidents by State

3. Detected MMR Genes in Lynch Syndrome Families Lynch Syndrome (HNPCC) 80% develop colorectal cancer other cancers include: kidney, stomach, ovary, small bowel, pancreas, & bile duct Mismatch Repair Deficiency Detected Mutations (%) MMR Genes

4. DNA Repair Mechanisms - Mismatch Repair (MMR) DNA Damage Environmental (carcinogens, UV light) Metabolic activities (free radicals, replication) DNA Mismatch Repair Evolutionarily conserved process Fidelity of DNA replication a) Base substitution, insertion, and deletion mismatches and loops b) DNA lesions - environmental and intracellular stress c) Apoptosis MMR loss - multistage carcinogenesis

5. PROKARYOTIC MMR CH 3 mismatch MutS, MutL, MutH CH 3 mismatch CH 3 mismatch 5’ 3’ 5’ nick 3’ nick Exonuclease - ExoVII or RecJ Exonuclease - ExoI HelicaseII CH 3 5’ 3’ CH 3 5’ 3’ DNA Polymerase III DNA Ligase DNA Polymerase III CH 3 5’ 3’

6. MutL α – Understanding the Structure MLH1 – PMS2 ExoI MLH1 - functional domains

7. Research Question Compared to MLH1 wildtype and and non-pathogenic polymorphisms, how well does ExoI interact with certain MLH1 mutants? - L582V, K751R, L607H, and R755W - putatively associated with Lynch Syndrome - do not affect MLH1 protein stability -do not affect MLH1 - PMS2 interaction Hypothesis Compared to MLH1 wildtype and MLH1 non-pathogenic polymorphisms, L582V, K751R, L607H, and R755W will show a decreased ability in binding EXO1 - in vitro assays that measure interaction capabilities MLH1 - functional domains

8. Approach 1. Construct in vitro expression vectors containing coding regions to be expressed - MLH1 Wildtype - MLH1 Mutants: L582V, L607H, K751R, R755W - ExoI 2. Find a way to probe for ExoI -express protein and test antibody 3. Perform in vitro co-immunoprecipitation assays with ExoI, PMS2, and MLH1 variants

9. Plasmid Construction Excision of cDNA by restriction digestion Gel isolation of cDNA hMLH1 wt Ligation into linear pCite vector pCite Restriction digests (screening) and sequencing pCMV hMLH1 wt hMLH1 mutant hMLH1 mutant hMLH1 mutant

10. Constructing the Plasmid (Three Way Ligation) Isolate Fragments MLH1(part) MLH1(part containing mutation) hMLH1 wt hMLH1 mutant pCite hMLH1 mutant

11. Plasmid Screening pCite hMLH1 mutant restriction enzyme (Xho1) hMLH1 mutant hMLH1 2.4 kb 3.8 kb Restriction Digests ladder 1 2 3* 4 5 6* 7 8 9 10 11 12 13 14* 2000 kb 2500 kb 3000 kb 4000 kb 5000 kb 6000 kb 10,000 kb 15 16 17* 18* 19 20 21* 22* 23* 24 ladder 2000 kb 10,000 kb 2500 kb 3000 kb 4000 kb 5000 kb 6000 kb K751WR755W L582VL607H

12. Co-immunoprecipitaton Transcription Translation hMLH1 pCite hEXO1 PMS2 pCITE Antibody – PMS2 Antibody Binding Beads Co- immunoprecipitation WashWestern Blot pCITE

13. Example Co-immunoprecipitation Qualitative Measurement

14. Exonuclease Detection hEXO1 pCITE Transcription Translation protein Exonuclease ≈ 105 kD MCF-7 30ul 15ul 10ul 5ul MCF-7 30ul 15ul 10ul 5ul 250 150 100 75 50 U.S Biological (mouse monoclonal Ab) NeoMarkers (mouse monoclonal Ab) Questions 1.Is protein being produced? 2.Is there a problem with the detection method?

15. Flagging hEXO1 Octapeptide - DYKDDDDK Polymerase Chain Reaction (PCR) hEXO1

16. Flag-Exonuclease Detection 250 150 100 75 50 Dr. Binghui Shen City of Hope National Medical Center and Beckman Research Institute Flag-Exonuclease 30ul 15ul 10ul 5ul Exo 30ul 15ul 10ul Exo 15mg protein 4.5ng 2.2ng 1.5ng.75ng 1.5ng rabbit polyclonal anti-Flag Anti-Flag 4.5ng 2.2ng 1.5ng 1.5ng

17. Summary Insufficient antibody sensitivity to ExoI with two different mouse monoclonal Ab’s Constructed prokaryotic transcription vectors For MLH1 WT, MLH1 mutants, ExoI Added flag peptide to amino terminal of ExoI reading frame Insufficient antibody sensitivity to ExoI with Anti-Flag

18. Future Studies 1.FLAG carboxyl end of hEXO1 2.Try a different epitope tag 3. 35 S labeled Methionine 1 Schmutte, C., M. M. Sadoff, S. Guerrette, S. Acharya, and R. Fishel. Interactions of the human exonuclease I with DNA mismatch repair proteins hMSH2, hMSH3 and hMLH1. J. Biol. Chem., in press. 2 Tran, P. T., J. A. Simon, and R. M. Liskay. Interaction of EXO1 with components of MutLα in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, in press. 4. 1 Glutathione-S-transferase protein-protein interaction assay - GST fusion protein (PMS2) - 35 S labeled ExoI 5. 2 Two Hybrid Assay - PMS2 fused to a binding domain - ExoI fused to activation domain

19. Thank You! The Buermeyer Lab Dr. Andrew Buermeyer Dr. Scott Nelson Howard Hughes Medical Institute Undergraduate Research, Innovation, Scholarship & Creativity (URISC) Dr. Kevin Ahern

20. CTD NTD

21. CTD NTD Exonuclease

24. DNA Mismatch Repair (MMR)

25. 2) The PMS2 mutants will show a decreased ability to bind MLH1 compared to PMS wildtype 2) The PMS2 mutants will show a decreased ability to bind MLH1 compared to PMS wildtype Perform Ligations Using T4 DNA Ligase

27. Schmutte, C., M. M. Sadoff, S. Guerrette, S. Acharya, and R. Fishel. Interactions of the human exonuclease I with DNA mismatch repair proteins hMSH2, hMSH3 and hMLH1. J. Biol. Chem., in press. 74a. Tran, P. T., J. A. Simon, and R. M. Liskay. Interaction of EXO1 with components of MutLα in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, in press.