1 Sister chromatid cohesion is mediated by the cohesin complex, which also plays key roles in diverse biological 2 pathways including DNA replication restart, and DNA double-strand break (DSB) repair under genotoxic stress. 3 In vertebrates, the core cohesin complex consists of a tripartite ring assembled from SMC1, SMC3, RAD21, 4 and the fourth subunit SA1 or SA2. Germline mutations in core cohesin subunits lead to a wide spectrum of 5 human diseases collectively called ?cohesinopathies, and to increased incidence of cancers. Our current 6 understanding of cohesin functions is based on models in which cohesin DNA binding is mediated exclusively 7 through nonspecific entrapment of dsDNA by the cohesin ring. However, these models cannot explain 8 observations from numerous studies demonstrating the localization of the cohesin complex at specific 9 structures and sequences. We recently demonstrated that SA1 binds specifically to double-stranded telomeric 10 DNA. In addition, we found that SA2 displays high binding specificities for ssDNA gaps and DNA with 11 secondary structures (overhang, flap and fork). These new results raise fundamental questions regarding the 12 structure and dynamics of SA1/2-DNA complexes and their roles in preserving genome integrity. We 13 hypothesize that DNA sequence and structure-dependent DNA binding by cohesin SA1/2 preserve 14 genome integrity under genotoxic stress. We will test this hypothesis in the following ways. First, using both 15 bulk assays and single-molecule atomic force and real-time fluorescence microscopy imaging of proteins on 16 DNA, we will identify the domains on SA1/2 that mediate DNA binding, and the impact of phosphorylation and 17 nucleosomes on their DNA binding dynamics. Second, the mechanism underlying the unique cohesin-ring 18 independent SA1-mediated sister telomere cohesion process is still unknown. We will test the ?multi-site SA1- 19 TRF1-TIN2-mediated DNA-DNA bridging? model for sister telomere cohesion. We will define the architecture of 20 the telomeric cohesin complex by using a novel electrostatic force microscopy imaging technique to reveal 21 DNA paths within the assembled protein structure. To study the sequential steps in protein recruitment and 22 DNA-DNA bridging mediated by SA1-TRF1-TIN2 protein complexes, we will use real-time fluorescence 23 microscopy imaging of DNA molecules confined to nanochannels in microfluidic devices. Finally, to determine 24 roles for SA1/2 in preventing DNA damage-induced genome and telomere instability, we will investigate the 25 impact of SA1/2 DNA binding or phosphorylation mutants and protein depletion on the efficiency of joining 26 distal DNA ends during DNA DSB repair in cultured cells. Furthermore, we hypothesize that SA1-mediated 27 telomere cohesion prevents DNA damage-induced telomere instability. To explore this postulate, we will 28 examine how SA1 DNA binding and phosphorylation mutants and protein depletion impact telomere integrity 29 after cellular exposure to genotoxic agents that damage telomeres. These results will greatly advance our 30 understanding of the cohesin function in diverse genome maintenance pathways. 31 32 33 34 35 36 37 38 39 40