1. Measuring the persistence of MutS at a mismatch site after binding Nikki O’Donnell August 25, 2005 Department of Environmental and Molecular Toxicology Mentors: Dr. John Hays and Pete Hoffman

2. DNA Repair Systems One major concern for an organism’s survival and fecundity is genomic stability Errors in DNA can occur during synthesis or post replication from environmental factors (i.e. UV radiation) This is a major problem because errors lead to mutations, which can lead to a variety of problems including cancer Organisms have multiple repair systems –Direct Reversal –Base Excision –Recombination –Nucleotide Excision –Mismatch Repair

3. Relevance of Mismatch Repair Evolutionary conserved process Essential for correcting errors during DNA replication Increases genomic stability 100 to 1,000 fold Lack of MMR has been linked to several forms of cancer

4. Overview  Relevance  Outline of Mismatch Repair  Question and Hypothesis  Project Design Objectives Objectives  Project Methodology Experimental Setup Experimental Setup Experimental protocol Experimental protocol Mutant Purification Mutant Purification Assay Validation Assay Validation  Key Findings / Results  Acknowledgements

5. DNA Mismatch Repair in Prokaryotes

6. Question and Hypothesis Question Question Does MutS stay fixed at a mismatch site after binding or move away? Does MutS stay fixed at a mismatch site after binding or move away? Hypothesis Hypothesis MutS remains near the mismatch site and does not migrate MutS remains near the mismatch site and does not migrate

7. Experiment Setup Wild Type MutS MutS stays at the mismatch site DNA is cleaved G G A T C C C C T A G G G G A T C C C C T A G G G G A T C C C C T A G G

8. Project Designs

9. Experiment Protocol Start with an E.coli plasmid with two specific single stranded nicking sites Start with an E.coli plasmid with two specific single stranded nicking sites Heat to denature bonds Heat to denature bonds Make oligomer and run the reaction with a complementary strand Make oligomer and run the reaction with a complementary strand ‘Gapped Plasmid’ ‘Gapped Plasmid’ Introduce new oligomer Introduce new oligomer Ligate Ligate Final product is plasmid with a mismatched base pair Final product is plasmid with a mismatched base pair

10. Mutant MutS Protein Mutation found in yeast made the mutS protein non responsive to ATP Testing in E.coli MutS protein. Validate by sequencing, expression of the protein and analysis of its biochemical characteristics * Specific site was found by another lab and was published in a paper. Strand Synthesis 1)Thermal cycle to denature DNA template 2)Anneal mutagenic primers containing desired mutation 3)Extend primers with PfuUltra DNA polymerase 4) Digest Parental DNA 5) Transform mutated molecule into competent cells for nick repair 1 2 3 4 5

11.  Induction in E.coli  Samples taken at hour intervals to check for expression  Check for correct size Protein expression over time 1 2 3 4 5

12. Assay Validation  Two restriction sites- one near and one farther removed from the mismatch-- BAMHI site  Test binding and protection  Do this in quick time and add BAMH1  Determine if cleavage happens

13. Key findings and Results Possible outcomes Separate wild type and mutant on a gel to separate protected DNA and possible cleaved segments DNA shift indication uncut Cut Time

14. Acknowledgements The Howard Hughes Medical Institute Dr. Kevin Ahern Dr. John Hays Pete Hoffman The Hays Lab Crew: Gerrick, Laurel, Aly, Mark, Stephanie, Huixian