Our genome encodes critical information that is required for the healthy function of every cell, tissue, and organ. However, genomic DNA is continuously accumulating toxic damage that arises during normal cellular processes, or is caused by environmental conditions such as sunlight and chemical carcinogens. Double- stranded DNA breaks (DSBs) are the most dangerous lesions. DSBs occur when both strands of the DNA double helix are broken in close proximity to each other, fragmenting the chromosome into two distinct pieces. If unrepaired, even a single DSB can initiate cellular dysfunction, malignant transformation, and tumor growth. Our cells can repair DSBs via two distinct pathways: error-prone non-homologous end joining or high-fidelity homologous recombination. Remarkably, the primary molecular steps that determine the DNA repair pathway are still not completely known. Thus, there is a critical need to understand how healthy cells repair their fragmented DNA and how disruptions in these processes can lead to cancer. Our long-term goal is to understand how specialized DNA repair proteins serve as the molecular caretakers of the genome. To achieve this goal, we pioneered a unique in vitro microscopy technique that can image multiple enzymes and record their biochemical activities as they repair DNA in real time. Using this technique, the Aims in this proposal will investigate how a group of human enzymes coordinate the first steps of DSB repair. First, we will determine how the Mre11/Rad50/Nbs1 (MRN) complex acts as the molecular sensor for DSBs in the context of chromatin. Second, we will investigate how MRN recruits additional enzymes to the DSB, and how these enzymes process a nucleosome-coated DNA track. Third, we will determine how MRN directs repair towards the homologous recombination pathway. In sum, our studies will elucidate the first critical steps of DSB repair and answer the long-standing question of how these enzymes biochemically define the DSB repair pathway. Ultimately, this knowledge will be required for developing new diagnostics and therapeutics that specifically target cancer cells that have lost the ability to correctly repair their genomes.
Each of our cells must constantly locate and repair DNA damage that is an unavoidable consequence of cellular metabolism. We will discover how key human DNA repair proteins locate and begin repairing DNA breaks, the most toxic forms of damaged DNA. Our anticipated findings will yield a more complete understanding of human DNA break repair and how dysfunction in any of these proteins leads to genetic instability and cancer.
| Bhattacharyya, Sudipta; Soniat, Michael M; Walker, David et al. (2018) Phage Mu Gam protein promotes NHEJ in concert with Escherichia coli ligase. Proc Natl Acad Sci U S A 115:E11614-E11622 |
| Jones Jr, Stephen K; Spivey, Eric C; Rybarski, James R et al. (2018) A Microfluidic Device for Massively Parallel, Whole-lifespan Imaging of Single Fission Yeast Cells. Bio Protoc 8: |
| Hawkins, John A; Jones Jr, Stephen K; Finkelstein, Ilya J et al. (2018) Indel-correcting DNA barcodes for high-throughput sequencing. Proc Natl Acad Sci U S A 115:E6217-E6226 |
| 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 |
| Myler, Logan R; Finkelstein, Ilya J (2017) Eukaryotic resectosomes: A single-molecule perspective. Prog Biophys Mol Biol 127:119-129 |
| 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; 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 |
| Jung, Cheulhee; Hawkins, John A; Jones Jr, Stephen K et al. (2017) Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips. Cell 170:35-47.e13 |
| 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 |
