Maintaining genetic integrity is crucial for cell viability and disease suppression. Consequently, cellular machinery such as the DNA damage response (DDR) has evolved to combat continual genotoxic insults. Whilst specific DNA repair mechanisms have been defined in great detail, understanding the spatiotemporal regulation of DDR factors in the cell remains a key challenge. Here, the covalent postranslational modifiers (PTMs) SUMO and ubiquitin play critical roles, both recruiting DDR proteins to DNA lesions, and then removing them as necessary to promote repair. Indeed, defects in the SUMO and ubiquitin pathways cause failed DDR orchestration, severe genetic instability, and disease. Therefore, the overarching goal of our research is to delineate SUMO and ubiquitin mediated mechanisms that maintain genome integrity, with an eye to identifying and exploiting potential therapeutic avenues. Our proposal centers on two factors, STUbL and SMC5/6, which integrate signaling through SUMO and ubiquitin to support key health-related processes. Of note, STUbL mediates the therapeutic effects of arsenic trioxide in leukemia, and SMC5/6 mutations cause severe disease. STUbL is an E3 ubiquitin ligase that selectively recognizes and ubiquitinates SUMOylated proteins to promote their degradation and/or extraction from chromatin. SMC5/6 is functionally related to cohesin and condensin but uniquely, can modify targets with SUMO and ubiquitin. To provide functional insights, we used proximity labeling to reliably map the proteomic environments of STUbL and SMC5/6 in key health-related settings such as: dysfunctional telomeres, DNA repair, and viral replication. Surprisingly, considerable overlap was identified between each proteome, creating further synergy and efficiency in our research. For example, the functions and targets of SMC5/6 and STUbL intersect in the “alternative lengthening of telomeres” (ALT) pathway used in ~15% of cancers. Thus, we would define STUbL and SMC5/6 roles in the elongation and “trimming” of telomeres through the novel ALT-specific targets and cofactors we identified. In addition, a wealth of recent data supports our hypothesis that STUbL controls SUMO pathway homeostasis, as well as specific targets, to support genome stability, DNA replication, and cell survival. Further analysis using genetic manipulation of the SUMO pathway (e.g. CRISPR/Cas9), mediators of SUMO chain toxicity, and new STUbL targets would establish this key paradigm in SUMO and ubiquitin pathway crosstalk. We also recently identified an SMC5/6 cofactor that binds SUMO and directs the complex to phase-separated ALT PML nuclear bodies, sites of viral replication, and likely DNA lesions, thereby unifying these seemingly disparate processes. Hence, we would define functions for SMC5/6 and its new SUMO binding cofactor in each of these processes to reveal common mechanisms. Overall, our collaborative teams' analysis of STUbL and SMC5/6 using proteomic, genetic, cell biolog...