In vitro reconstitution of the ubiquitylation and disassembly of the eukaryotic replisome
Maintenance of genomic integrity is dependent on the duplication of chromosomes, only once per cell cycle. Highly conserved mechanisms for the regulation of chromosome replication exists to ensure that the genome is copied only once. The Cdc45-MCM-GINS (CMG) DNA helicase which is the core of the eukaryotic replication complex, has been shown to be extensively regulated by post translational modifications, during its assembly. Therefore, it is not inconceivable that the process to unload the replication complex would also be a conserved and regulated process. In 2014, our lab discovered that the CMG complex undergoes post-translational modification in the form of ubiquitylation on one of the subunits of CMG, leading to its disassembly from the chromatin. Though the main players in the disassembly of CMG were known, viz the E3 ligase SCFDia2 and segregase Cdc48, very little was known about the mechanism of CMG disassembly. In the process of learning more about the disassembly of the replicative helicase from chromatin, I reconstituted the ubiquitylation of CMG and thereafter the disassembly of CMG helicase in vitro. My work resulting in the reconstitution of CMG disassembly in vitro is the first example of the disassembly of a multi-subunit physiological substrate of Cdc48. Though CMG is ubiquitylated in yeast extracts in vitro, it does not lead to its disassembly and therefore led me to find conditions necessary for the efficient ubiquitylation of CMG. I have further shown that purifying the E3 ligase associated CMG can be efficiently ubiquitylated in a semi-reconstituted system consisting of purified factors, necessary for the ubiquitylation of substrate. I investigated whether this efficiently ubiquitylated CMG can be disassembled by purified Cdc48 and associated co-factor Ufd1/Npl4 in vitro and found that disassembly is dependent on K48 linked poly-ubiquitylation of CMG. I have found that the reconstituted poly-ubiquitylation of CMG is restricted to the Mcm7 subunit of CMG, recapitulating the ubiquitylation of CMG in vivo, and my data points out that there are multiple sites of ubiquitylation on Mcm7. Through this work, I have also found that ubiquitylated Mcm7 no longer associates with the rest of the CMG components after disassembly of CMG. My assays and findings, open the door towards dissecting the molecular mechanism of the disassembly of CMG in greater detail.
Disassembly, Ubiquitin, CMG, Replisome, ubiquitylation, Cdc48, p97, segregase, SCF ligase, Cullin ligase
Networked Digital Library of Theses and Dissertations