The Role of Mismatch-Repair Protein MLH3 in Genomic Stability in the Plant Arabidopsis Howard Hughes Medical Institute Summer Research Program Laurel Wheeler Mentor: Dr. John Hays

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

Mismatch Repair (MMR) MMR is responsible for locating and removing mismatched base pairs Highly conserved throughout evolution Increases genomic stability 100 to 1,000 fold Lack of MMR has been linked to several forms of cancer

Recognition of Daughter Strand MMR telling the difference between daughter and parent stands is critical in repair In prokaryotes MMR recognizes the difference between the two strands by looking for the unmethylated strand A stand is methylated shortly following replication, thus the unmethylated strand is the daughter stand

Prokaryotic MMR Mismatch is recognized MutS binds to DNA at mismatch site MutL recruited by MutS MutL acts as matchmaker between MutS and MutH Recognizes correct strand because it is unmethylated MutH creates a nick 5’ to the unmethylated GATC site Exonuclease degrades section of the strand containing mismatch Gap in DNA is resynthesized cmgm.stanford.edu/biochem201/Slides/DNA%20Repair/24%20mismatch%20r

Eukaryotic MMR Instead of MutS, MutL, and MutH, there are several MutS and MutL homologues MutS and MutL homologues exist in heterodimers

Arabidopsis thaliana Investigation of the MLH3 homologue found in Arabidopsis plants

MLH3 ??? Unknown function of MLH3 Use MLH3 knockout mutants to look for irregularities in plants lacking this part of MMR Possible problems with meiosis? Sterility? Phenotypic changes?

Current Experiments Underway Look for MLH3 knockout homozygous lines Identify homozygotes through genotyping Confirm through MSI assays

PCR using primers LBa1 and MLH3-R PCR using primers MLH3-F and MLH3-R Genotyping the Plants

MMR Correction of Slip-Mispairing AT NNNATATAT ATATAT NNNTATATA TATATATATATANNN NNNATATATATATAT NNNTATATATATATATATATANNN NNNATATAT ATATAT NNNTATATA TATATATATATANNN TA +2 base insertion no insertion or deletion -2 base deletion ReplicationMMR MMR

Microsatellite Instability (MSI) PCR was run using primers specific for microsatellite sites PCR products were further examined by the ABI 3100 capillary electrophoresis analyzer ABI prism software was used to interpret data

This work was supported by a grant from the Howard Hughes Medical Institute As well as in part by the Dr. John Hays’ lab Funding of the Project

Acknowledgements Dr. John Hays for acting as a mentor throughout the research process Adela Torres, Peter Hoffman, Stephanie Bollmann, and Dr. Huixian Wang for training and guidance