Genetic and molecular dissection of the role of dissolution and resolution in the maintenance of genomic stability

Resumen

DNA is constantly under the assault of many kinds of damage, both of exogenous and endogenous origin. The very replication of DNA molecules is a major source of this second kind of injury, for its impairment can generate a panel of toxic intermediates, where both sister chromatids are linked through DNA linkages. These structures are constantly monitored and cleared off in cells by two independent pathways, denominated dissolution and resolution. While the latter is catalyzed by structure-specific nucleases including MUS81, GEN1 and SLX1/SLX4, dissolution is known to be operated through the coordinated action of the helicase BLM and the topoisomerase TOPIII¿. A wealth of data from lower eukaryotes suggest that a complex known as SMC5/6, related to condensin and cohesin, might also be involved in dissolution ¿ yet its contribution along this pathway in mammals is largely uncharacterized. We here used mouse models to explore a potential role of the SMC5/6 complex in dissolution in mammals. For this purpose, we employed a recently published Smc6 mutant allele, as well as a new allele that carries a mutation on Nsmce2, a SUMO ligase which is an essential functional component of the SMC5/6 complex. Our data show that the Nsmce2SUMOdead allele generated is indeed severely compromised in its SUMO ligase activity. Surprisingly, whereas some signs of genomic instability can be detected in Nsmce2 mutants in vitro, our results suggests that NSMCE2-dependent SUMOylation is largely dispensable for fitness in vivo. In addition, we find a severe synthetic lethality between the resolution resolvase MUS81, and the Smc6-impairing mutation we employed. These results help to substantiate a role for SMC5/6 in dissolution in mammals, and to dimension the role that SUMOylation might play within the SMC5/6 complex. Besides dissolution, this work explored two aspects of the resolution pathway - namely, a potential role for resolution nucleases on the phenomenon of ¿chromosome pulverization¿, and the implementation of cell-systems where the activity of these nucleases can be unleashed at will. Collectively, my PhD tried to explore the concepts of resolution and dissolution of joint DNA molecules, something that has mostly been investigated using yeast as a model system.