Integrative single molecule studies: DNA repair and technology development

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Project Summary The overall objective of this proposal is to use existing and develop new 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 DNA. The transient nature of these interactions presents significant challenges to 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. My laboratory focuses on elucidating the molecular mechanisms of DNA mismatch repair (MMR) and on development of single-molecule tools that give us access to previously unattainable information. MMR plays a major role in reducing genomic mutations, 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, as well as resistance to several DNA damaging agents used to treat cancer. Surprisingly, in trinucleotide repeat (TNR) expansion, which causes some neurodegenerative diseases, MMR proteins cause mutations that promote repeat expansion and disease. MutSa/MutSb initiates MMR by binding to a mismatch/insertion deletion loop and undergoing ATP-dependent conformational changes that promote its interaction with one or more MutLa proteins. Subsequently, PCNA and ATP activate MutLa to incise the daughter strand, and MutSa activates EXO1 to processively excise the DNA containing the error, followed by resynthesis and ligation. Similarly, in TRE, MutSb binds looped out DNA trinucleotide repeats and recruits MutLa and/or MutLg, but instead of leading to repair, this process promotes expansions. Understanding the molecular mechanisms that underlie these different processes is essential for developing effective treatments for the associated cancers and neurodegenerative diseases. 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 and TRN expansion. We will continue our mechanistic studies of MMR and extend them to TNR expansion. We are taking an integrative approach in which we utilize an array of single-molecule techniques, including AFM and single-molecule fluorescence, to examine MMR in multiple organisms in vitro and in vivo, as well as initiating studies on TNR expansion. We will focus on examining the temporal and spatial assembly of proteins on DNA during initiation of MMR and TNR expansion. Finally, we will continue to develop single-molecule tools, such as DREEM that allows us to “see” DNA inside protein-DNA complexes and “high-throughput” AFM. These technologies expand the single- molecule toolbox, enrich the structural information from AFM, and directly benefit our MMR studies.