Title: Functional Studies of Individual DNA Repair Enzymes Using Single Molecule Fluorescence

Abstract: Enzymes are protein machines that can undergo drastic structural rearrangements in response to specific molecular interactions. These conformational changes and interactions are critical to initiating, regulating, and terminating the diversity of pathways in living systems that are responsible for maintaining life. Mutations in DNA can lead to amino acid substitutions in proteins that impact enzyme structure and function. Although genetic mutations are linked to many diseases such as cancer, we are often unaware of how the resulting amino acid substitutions directly impact the molecular behavior of the enzymes. I will describe the use of in vitro single molecule fluorescence resonance energy transfer (smFRET) to investigate the molecular function of individual DNA repair enzymes. Specifically, I will focus on the coordinated functions of protein machines that act on DNA mismatches. DNA mismatch repair (MMR) is a post-replicative pathway that corrects rare mistakes in the genome of all organisms. MMR is initiated by MutS and MutL homologs which contain DNA binding and ATPase activity. Single amino acid mutations in MutS and MutL enzymes that presumably cause failures in molecular function have been linked to hereditary and sporadic colorectal cancer. A fundamental understanding of the MMR process is necessary to uncover how these failures likely initiate the formation of tumors. Using site-specific fluorescence labeling strategies with smFRET, we have identified sequential pathways of conformational changes in MutS and DNA that are likely necessary to initiate the MMR pathway. Understanding dynamic protein-protein and protein-DNA interactions represents a key step toward pinpointing failures in mutants and elucidating molecular mechanisms of cancer development. Extending investigations of similar enzymatic pathways linked to cancers into in vivo systems represents a next phase of research.