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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Transition Award (R00)
Project #
4R00GM097177-03
Application #
8616481
Study Section
Special Emphasis Panel (NSS)
Program Officer
Preusch, Peter C
Project Start
2011-04-05
Project End
2016-02-29
Budget Start
2013-03-14
Budget End
2014-02-28
Support Year
3
Fiscal Year
2013
Total Cost
$248,999
Indirect Cost
$16,667
Name
University of Texas Austin
Department
Type
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78712
Robison, Aaron D; Finkelstein, Ilya J (2014) High-throughput single-molecule studies of protein-DNA interactions. FEBS Lett 588:3539-46