Cryo-EM snapshots of CMG helicase assembly
File(s)
Author(s)
Pühringer, Thomas
Type
Thesis
Abstract
The accurate, complete and timely replication of DNA is essential for the propagation of life. Every proliferating cell needs to duplicate its entire genome before it can divide. At the heart of this process lies the replicative helicase MCM, which unwinds parental DNA, thereby providing the single-stranded template for replicative polymerases. In eukaryotes, DNA replication initiates at multiple sites along their chromosomes, each replication unit must only be replicated once per
cell division. To prevent cancer-causing re-replication, the loading and activation of the replicative helicase is regulated through the cell cycle. From late mitosis to G1 phase, MCM is loaded as an inactive double hexamer at replication origins, it cannot unwind DNA yet. In S phase, two kinases, DDK and CDK, drive the recruitment of two helicase activator, Cdc45 and GINS, to MCM, assembling the replicative CMG helicase. While we know all molecular players essential for CMG formation in yeast, we do not understand the
structural mechanisms that regulate the faithful assembly of CMG. Hence, I combined biochemical reconstitution and cryo-electron microscopy to determine structures of CMG assembly intermediates. My work reveals that the CMG assembly factor Sld3 recognises a conformational change within the MCM double hexamer upon DDK phosphorylation, delivering Cdc45 to MCM. By establishing CDK-bypass reagents, I reconstituted the pre-initiation complex and determined its cryo-EM structure. Upon pre-initiation complex formation, the assembly factor Dpb11 splays the two MCM rings apart and recruits the activator GINS. ATP binding matures the pre-initiation complex into the CMG helicase. The structural transitions imposed during CMG maturation explain the eviction of multiple CMG assembly factors. Together, my thesis provides a mechanistic framework for the regulated assembly of the replicative CMG helicase.
cell division. To prevent cancer-causing re-replication, the loading and activation of the replicative helicase is regulated through the cell cycle. From late mitosis to G1 phase, MCM is loaded as an inactive double hexamer at replication origins, it cannot unwind DNA yet. In S phase, two kinases, DDK and CDK, drive the recruitment of two helicase activator, Cdc45 and GINS, to MCM, assembling the replicative CMG helicase. While we know all molecular players essential for CMG formation in yeast, we do not understand the
structural mechanisms that regulate the faithful assembly of CMG. Hence, I combined biochemical reconstitution and cryo-electron microscopy to determine structures of CMG assembly intermediates. My work reveals that the CMG assembly factor Sld3 recognises a conformational change within the MCM double hexamer upon DDK phosphorylation, delivering Cdc45 to MCM. By establishing CDK-bypass reagents, I reconstituted the pre-initiation complex and determined its cryo-EM structure. Upon pre-initiation complex formation, the assembly factor Dpb11 splays the two MCM rings apart and recruits the activator GINS. ATP binding matures the pre-initiation complex into the CMG helicase. The structural transitions imposed during CMG maturation explain the eviction of multiple CMG assembly factors. Together, my thesis provides a mechanistic framework for the regulated assembly of the replicative CMG helicase.
Version
Open Access
Date Issued
2024-10-18
Date Awarded
01/02/2025
License URL
Advisor
Costa, Alessandro
Sponsor
Francis Crick Institute
Boehringer Ingelheim Pharmaceuticals
Wellcome Trust (London, England)
Medical Research Council (Great Britain)
Cancer Research UK
European Research Council
Publisher Department
Department of Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)