ABSTRACT The single-stranded DNA binding protein (SSB) is the founding member of the nineteen-partner SSB interactome. There is a fundamental gap in understanding the mechanism of action of the interactome, the first prokaryotic family of oligosaccharide/oligonucleotide binding fold (OB-fold) genome guardians identified. The continued existence of this gap represents an important problem because, until it is filled, a complete and clear understanding of genome stability will be lacking. This understanding is crucial as defects in OB-fold genome guardian family members have disastrous consequences for genome stability. In higher organisms, mutations in the three OB-folds of BRCA2 ultimately result in cancer, and the proposed studies are therefore directly relevant to human disease. Consequently, the long-term goal is to understand the molecular mechanism of the SSB interactome. The main objective of this proposal is to understand how SSB interacts with, and regulates, several partner proteins and itself, to maintain genome integrity. The central hypothesis is that two regions of SSB known as the linker and the OB-fold, (present in both SSB and interactome partners), are the key elements to all aspects of protein function. The rationale for the proposed research is that once it is known how SSB physically and functionally interacts with both itself and its partners, a clearer understanding of the events required to maintain genome integrity, mediated by this OB-fold family of genome guardians, will be obtained. The central hypothesis will be tested by pursuing two specific aims: 1) determine the role of the linker/OB-fold network in SSB function; and 2) determine the mechanism of action of the SSB interactome. Under the first aim, a combination of in vivo and in vitro binding assays, HDX-MS, protein crystallography, cryo-EM, and single-molecule biochemistry will be used to determine whether the linker/OB-fold interface is the primary means of SSB-partner binding. Under the second aim, enzyme kinetics, genetics, real-time super-resolution microscopy, and single-molecule biochemistry will be used to understand how the linker/OB- fold network mediates SSB interactome function in the dynamic reactions of DNA replication, recombination, and repair. The proposed research is innovative because of the combinatorial strategy taken, the novel single-molecule approaches used, and the care that we will take in elucidating SSB interactome function using full-length proteins. The proposed research is significant because it will allow, for the first time, the development of a clear picture of SSB interactome function in DNA metabolism and the maintenance of genome integrity.