ROLES OF THE SMC5/6 COMPLEX IN DNA REPAIR AND CHROMOSOME INTEGRITY IN VERTEBRATE CELLS

DNA damage response (DDR) mechanisms are crucial in all organisms to detect DNA damage and integrate its repair with cell cycle control, chromatin structure, and other adaptive changes in cell physiology. The highly conserved structural maintenance of chromosomes (SMC) complex, SMC5/6, contains both SUMO and ubiquitin ligase activities in its composition and is crucial for processing recombination structures arising during replication. Differently from related SMC complexes such as Cohesin and Condensin, the function of SMC5/6 in genome maintenance and its mode of action remain to date hardly understood. Moreover, a lack of cellular models for SMC5/6, especially in human cell lines, has hampered progress in understanding its roles in DDR in model systems most relevant for human disease. Here, we established vertebrate and human cellular models of SMC5/6 knockouts and characterized the associated phenotypes in a battery of assays. Specifically, I established (conditional) SMC5 knockout (KO) in chicken (DT40) and human (TK6) B cell lines. We generated a conditional SMC5 KO in TK6 human cell lines, and uncovered that SMC5 depletion caused apoptosis-mediated lethality, associated with increased DSBs and cohesion defects. In chicken DT40 cell lines, SMC5 KO causes a slow growth phenotype depending on the temperature condition. Second, we investigated the roles of SMC5/6 in intra- and inter-strand crosslink (ICL) repair. SMC5 shows epistasis with FA core mutations, as well as with DDX11 and RAD17, without affecting FANCD2-I ubiquitination and CHK1 phosphorylation. We propose that SMC5 is working downstream of FA and RAD17/DDX11-mediated pathways, likely in resolving the emerging recombination intermediates. Third, we are attempting to identify the principles underlying SMC5/6 roles in the establishment/maintenance of chromatin structure/cohesion and integrity of specific genomic loci such as replicating fragile sites and centromeric/peri-centromeric regions. To these ends, we have used BLISS, a genomic approach to map DSBs, and plan to employ ChIP-seq of endogenously tagged SMC5/6.