ABSTRACT Efficient genome maintenance is a double-edged sword. Accurate repair of DNA lesions and damaged replication forks promotes genome stability, which is a key to avoiding cancer, aging and neurodegenerative diseases associated with DNA repeat expansion. The same mechanisms that maintain genome integrity in healthy cells, allow cancer cells to acquire a more aggressive character and develop resistance to radiation and chemotherapy. Untimely deployment and/or dysregulation of the DNA repair machines may further destabilize the genome or may result in the accumulation of geno- and cytotoxic repair intermediates. Significant gaps remain in our understanding of the molecular events that funnel the intermediates of otherwise accurate repair into “rogue”, genome-destabilizing mechanisms. This research program emphasizes the molecular and structural mechanisms by which the intermediates of DNA metabolism bound by Replication Protein A (RPA) are channeled into DNA repair, protection of DNA replication forks, homologous recombination, DNA damage tolerance and signaling. Our central hypothesis is that the activities of the RAD51 recombinase, the ssDNA-binding protein RPA, recombination mediators BRCA2 (in human) and Rad52 (in yeast), and DNA repair helicases are finely tuned by a variety of factors, which include posttranslational modifications, interacting partner proteins, specific DNA structures and DNA lesions. These factors affect the protein configurational dynamics and critical protein-protein interfaces. Understanding how the protein plasticity and kinetics of assembly of the macromolecular machines of DNA repair will show us new ways to selectively manipulate the activities of RAD51, RPA and multifunctional DNA helicases and polymerases in DNA replication and repair. We are leveraging and building the tools of single-molecule biochemistry, biophysics, structural and chemical biology. Our unique perspective on the formation, activities and regulation of the nucleoprotein complexes orchestrating genome maintenance is rooted in our ability to visualize microscopic configurational dynamics of and sort individual human DNA repair proteins with their native posttranslational modifications, and to probe and separate activities associated with different surface-tethered proteins and nucleoprotein complexes at the single- molecule level. Our goal is to provide an entirely new outlook on how the cell balances the assembly and activities of the molecular machines that can repair, but also destabilize, the genome, and to be able to alter this balance with new chemotherapeutics targeting cancer and neurodegenerative diseases.