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|Title:||Defining a Role for the Smc5/6 Complex Component Nse5 in DNA Replication and DSB Repair|
|Abstract:||The Smc5/6 complex is a member of the Structural Maintenance of Chromosomes (SMC) family of proteins that provides support and organization to chromatin. While other SMC family complexes, such as cohesin and condensing, have each been extensively studied, relatively little is known about the function of the Smc5/6 complex. In addition to core heterodimer Smc5 and Smc6, six additional non-SMC elements (Nse1-6) exist in this complex, one of which (Nse2/Mms21) is an E3 small ubiqutitin modifier (SUMO) ligase. Here we investigate the multifaceted roles of the Smc5/6 complex in the face of genotoxic stress, and assess the contribution of the Nse5 component in facilitating these functions. We find that upon treatment of cells with replication inhibitor hydroxyurea, the Smc5/6 complex is recruited genome-wide to stalled replication forks and is crucial in preventing fork collapse. When fork collapse does occur, the Smc5/6 complex appears to be important in resolving Rad51-dependent X-shaped DNA structures, thought to be HR intermediates. Nse5 is crucial in maintaining Smc5/6 complex integrity, and in promoting SUMOylation of Smc5; however, in the face of replication stress, only Smc5/6 complex integrity is essential. Altogether our data indicate two distinct roles for the Smc5/6 complex at stalled forks: in preventing collapse, and in resolving toxic X-shaped DNA structures. We have additionally described a central role for the Smc5/6 complex in regulating chromatin dynamics following generation of an irreparable DSB. In the absence of functional Smc5/6 complex, movement to both the Nuclear Pore Complex (NPC) and envelope-bound Mps3 is completely impaired. We have narrowed down Smc5/6 complex function to two separate roles: Mms21-dependent SUMOylation at the break site, and Nse5-dependent recruitment of SUMO targeting ubiquitin ligase Slx5 to break sites. Additionally we have identified the signal that triggers break relocation as SUMOylation, with mono-SUMOylation triggering relocation to Mps3 and poly-SUMOylation triggering relocation to the NPC. Altogether our data describes novel roles for the Smc5/6 complex in maintaining genomic integrity during replication stress, and regulating chromatin dynamics during DSB repair. We propose that the Smc5/6 complex fulfills its functions through both physical tethering of chromatin, and Mms21-dependent signalling in response to DNA damage.|
|Appears in Collections:||Electronic Theses|
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