The mechanism of CMG helicase disassembly during the completion of eukaryotic chromosome replication

Abstract

To ensure that the duplication of chromosomes in eukaryotic cells can occur only once per cell cycle and to maintain genome integrity, the highly conserved process of eukaryotic chromosome replication employs different mechanisms of regulation. The Cdc45-MCM-GINS (CMG) DNA helicase, which is at the core of the eukaryotic replisome complex, has previously been shown to be extensively regulated by phosphorylation in the assembly of the helicase, but until now it has been unclear whether CMG is also controlled by other post-translational modifications such as the addition of small protein modifiers. I have found that the CMG helicase is modified in vitro in extracts of S-phase budding yeast cells by K48-linked polyubiquitylation. The in vitro ubiquitylation is restricted to the Mcm7 subunit and is highly specific, since it only occurs in the context of the CMG helicase complex. Since K48-linked polyubiquitin chains in vivo often target proteins to the proteasome, or to the ubiquitin-selective segregase known as Cdc48 in yeast and p97/VCP in animal cells, I investigated the consequences of inactivating the proteasome or Cdc48 in asynchronous cultures of yeast cells. Strikingly, inactivation of Cdc48 led to the accumulation in vivo of CMG complexes with ubiquitylated Mcm7, suggesting that ubiquitylation of CMG normally precedes Cdc48-dependent disassembly. Furthermore, in vivo ubiquitylation of CMG helicase was shown to be a late event in chromosome replication and is not inhibited by the S-phase checkpoint. In addition to that, I performed a screen with all E3 ubiquitin ligases known to regulate genome stability, which showed that in vivo ubiquitylation of Mcm7 was dependent upon SCFDia2. Cdc48, a novel factor in CMG helicase regulation, can specifically interact with the ubiquitylated CMG helicase. Moreover, the analysis of the timing of these events upon inactivation of Cdc48 during a single cell cycle showed that in vivo ubiquitylation of CMG helicase accumulates towards the completion of chromosome replication, while blocking the subsequent disassembly of the CMG helicase on chromatin.These findings identify an essential role for Cdc48 in disassembly of CMG when cells complete chromosome replication, indicating that disassembly of the CMG helicase is just as carefully regulated as the assembly step during the initiation of chromosome replication.

Date of Award	1 Aug 2015
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Iain Hagan (Supervisor) & Karim Labib (Supervisor)