The overall objective of this proposal is to develop and apply single-molecule techniques to gain mechanistic insights into the critical processes occurring during DNA repair. DNA repair processes, which are the guardian of the genome, involve multiple sequential enzymatic steps that require the coordinated assembly and action of many proteins on the DNA. The transient nature of these interactions presents significant challenges in elucidating the molecular mechanisms of DNA repair using traditional biochemical methods. Single molecule approaches are well suited to overcome these difficulties; however, they present their own challenges, requiring innovative solutions. Research in my laboratory focuses on elucidating the molecular mechanisms of DNA mismatch repair (MMR) and on the development of single-molecule tools that will give us access to previously unattainable information and/or greatly facilitate throughput of implementation or analysis. MMR plays a major role in mutation avoidance, including correcting DNA replication errors, modulating cellular responses to DNA damaging agents, and preventing recombination between diverged sequences. Mutations that inactivate MMR proteins cause Lynch syndrome, the most common hereditary cancer. In addition, they cause resistance to the cytotoxic effects of several DNA damaging agents that are often used in the treatment of cancer. As such, understanding the molecular mechanisms that underlie these different processes is essential for developing effective treatments for the associated cancers. MutS? initiates repair by binding to a mismatch and undergoing ATP-dependent conformational changes that promotes its interaction with one or more MutL? proteins. Subsequently, PCNA activates MutL? to incise the daughter strand in an ATP-dependent manner. Once MutL? nicks the DNA 5' to the mismatch, MutS? can activate the 5'-3' exonuclease EXO1 to processively excise the DNA containing the error or promote POL?/? to initiate strand-displacement synthesis. Finally, DNA polymerase ? or ? catalyzes resynthesis, and DNA ligase seals the nick. Single-molecule, structural, and biochemical studies, including several from our laboratory, indicate that the conformational dynamics and assembly states of the proteins and protein-DNA complexes are central to the regulation of MMR. We will extend our ongoing studies to decipher the molecular mechanisms critical to MMR. We are taking an integrative approach in which we utilize an array of single-molecule techniques to examine MMR in multiple organisms in vitro and in vivo. We will focus on examining the temporal and spatial assembly of MMR proteins on the DNA during MMR initiation. To further our (and others) ability probe these mechanisms, we will continue to develop new single-molecule tools, focusing on: 1) development of a high-throughput platform for preparation and imaging of AFM samples and 2) optimization of our newly invented electrostatic force imaging technique, called DREEM, that allows us to ?see? DNA inside protein-DNA complexes. In addition to expanding the single-molecule toolbox, the this technology will directly benefit our studies of MMR.
The goal of this grant is to develop single-molecule technologies and use them along with existing technologies to examine the molecular mechanism of DNA mismatch repair. DNA mismatch repair plays a major role in mutation avoidance, and mutations in mismatch repair genes dramatically increase the frequency of mutations and cause resistance to chemotherapy and the most common hereditary cancer. This research will provide needed tools to the research community and further our understanding of the molecular mechanisms that underlie mismatch repair, which will be important for designing treatment of cancers in which mismatch repair is compromised.