To maintain stable genomes, cells carry out an accurate and timely replication program and repair such
deleterious DNA lesions as double-stranded breaks, inter-strand crosslinks, and damaged replication forks.
Project 1 of the parent NIH R35GM131704 MIRA grant (PI: Spies) investigates the molecular machinery of
homologous recombination (HR), a cellular process that provides the most accurate means to repair of these
deleterious DNA lesions and damaged replication forks, and thereby contributes to genome stability in normal
cells, but also helps cancerous cells to develop resistance to radiation and DNA-damaging chemotherapy. We
are building a quantitative description of the central step in HR and its regulation, which will draw on the
importance of protein plasticity and conformational dynamics in molecular recognition. Project 2 investigates
multipurpose DNA repair helicases and their ability to coordinate DNA replication through difficult to replicate
regions, thus also contributing to genome stability.
Both projects utilize single-molecule total internal reflection fluorescence microscopy (smTIRFM), correlated
optical tweezers and fluorescence microscopy (CTFM), mass photometry and biochemical reconstitutions to
visualize and quantify the dynamic assembly and remodeling of the nucleoprotein complexes coordinating HR
and processing of alternative DNA structures. The key intermediate in all processes we study under the two
projects is a dynamic complex between ssDNA binding protein RPA (Replication Protein A) and DNA, including
ssDNA at resected DNA breaks, damaged replication forks, DNA repair intermediates, structures arising at DNA
repeats, and G-quadruplexes. Our single-molecule and biochemical data suggest that the architecture and the
dynamics of the RPA-DNA complexes is regulated by specific RPA partners and in differences in RPA
engagement to different DNAs. The structures and architectures of these complexes remain elusive.
Recent advances in CryoEM allowing us to advance a structural understanding of the RPA-DNA complexation
on unstructured and telomeric DNA. We are also pursuing structures of RPA-telomere-hnRNPA1 complex, and
FANCJ helicase bound to telomeric and cMyc G-quadruplexes. While we achieved a significant experimental
traction, data processing remains a bottle neck for our CryoEM work. This application requests funds for
acquisition of the Exxact workstation configured specifically for GPU accelerated CryoEM single particle 3D
reconstruction, which will allow us to consolidate in house data processing and structure determination. Progress
on the structures containing RPA and FANCJ complexes will help us to build a completely new picture of the
nexus between RPA configuraiotnal dynamics and shuttling of the RPA-containing complexes into specific
genome maintenance pathways.