Repairing broken DNA is essential for preventing mutations that can cause diseases such as cancer. Homologous recombination is an error-free DNA repair pathway that is conserved from bacteria to human. In the first step of recombination, Sgs1 and other specialized DNA motor proteins move along the broken DNA to process damaged strands for repair. Loss of Sgs1 in humans leads to devastating diseases such as Bloom, Werner and Rothmund-Thomson syndromes. The process by which Sgs1 and related DNA motors navigate on highly condensed chromatin and deal with other nucleoprotein collisions remains unresolved. Our hypothesis is that DNA motors collaborate to destabilize nucleosomes and other roadblocks by sequentially displacing and evicting the obstacles, thereby allowing other repair enzymes to gain access to the DNA. I have begun to address how DNA motors negotiate roadblocks by directly visualizing these collisions at the single molecule level. I observed that RecBCD, a prokaryotic DNA repair motor, displaces multiple types of obstacles as it moves along DNA. In the K99 phase, I will extend my single molecule assay to study the motor properties of Sgs1. During the R00 phase, I will elucidate the role of Top3/Rmi1 and Dna2 in facilitating Sgs1-dependent eukaryotic DNA repair.
My second aim i n the R00 phase is to determine how the Sgs1/Top3/Rmi1 complex processes chromatin. These experiments will rely on a new technology developed in the Greene laboratory, which allows us to directly visualize hundreds of individual DNA motor proteins in real time. By rapidly gathering statistically relevant datasets, we can study homologous recombination with an unprecedented level of mechanistic detail. My ultimate career goal is to achieve tenure as a professor at a research institution. The skills that I develop during the K99 phase of the fellowship will enable me to succeed as an independent investigator.
Breaks in DNA arise frequently as a result of external damaging agents and naturally during cell replication. This project aims to characterize a crucial DNA repair pathway used by cells to maintain genome stability. Understanding details of DNA repair mechanisms will shed light on various cancer-prone human diseases.
|Soniat, Michael M; Myler, Logan R; Schaub, Jeffrey M et al. (2017) Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination. Methods Enzymol 592:259-281|
|Myler, Logan R; Finkelstein, Ilya J (2017) Eukaryotic resectosomes: A single-molecule perspective. Prog Biophys Mol Biol 127:119-129|
|Myler, Logan R; Gallardo, Ignacio F; Soniat, Michael M et al. (2017) Single-Molecule Imaging Reveals How Mre11-Rad50-Nbs1 Initiates DNA Break Repair. Mol Cell 67:891-898.e4|
|Kim, Yoori; de la Torre, Armando; Leal, Andrew A et al. (2017) Efficient modification of ?-DNA substrates for single-molecule studies. Sci Rep 7:2071|
|Brown, Maxwell W; Kim, Yoori; Williams, Gregory M et al. (2016) Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions. Nat Commun 7:10607|
|Myler, Logan R; Gallardo, Ignacio F; Zhou, Yi et al. (2016) Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins. Proc Natl Acad Sci U S A 113:E1170-9|
|Gallardo, Ignacio F; Pasupathy, Praveenkumar; Brown, Maxwell et al. (2015) High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging. Langmuir 31:10310-7|
|Robison, Aaron D; Finkelstein, Ilya J (2014) Rapid prototyping of multichannel microfluidic devices for single-molecule DNA curtain imaging. Anal Chem 86:4157-63|
|Spivey, Eric C; Xhemalce, Blerta; Shear, Jason B et al. (2014) 3D-printed microfluidic microdissector for high-throughput studies of cellular aging. Anal Chem 86:7406-12|
|Robison, Aaron D; Finkelstein, Ilya J (2014) High-throughput single-molecule studies of protein-DNA interactions. FEBS Lett 588:3539-46|
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