Functional analysis of DNA replication initiation
File(s)
Author(s)
Barbon, Marta
Type
Thesis
Abstract
Initiation of DNA replication is highly conserved from yeast to human. A crucial
factor during this process is the replicative helicase MCM2-7, a ring-shaped
hetero-hexameric protein assembly, which unwinds DNA at the replication fork.
In a multi-step process, ORC, Cdc6 and Cdt1 promote the loading of two
MCM2-7 hexamer into a head-to-head double hexamer around double stranded
DNA.
The recent cryo-EM structure of an ORC/Cdc6/Cdt1/MCM2-7 (OCCM)
intermediate has revealed that the MCM2-7 hexamer and ORC/Cdc6 are
connected by C-terminal winged helix domains (WHDs). However, how these
WHDs function for helicase loading and MCM2-7 double-hexamer formation
has been largely unknown. Using a combination of biochemical and structural
approaches, we have investigated the function of the different Mcm C-termini
during pre-RC formation.
In this study, we developed a new concept, explaining how distinct
functions of the individual WHDs drive the multi-step pre-RC formation process.
In particular, we discovered that the Mcm5 WHD serves as a key factor in order
to signal helicase loading defects and to induce quality control during pre-RC
formation. In addition, it was found that the Mcm5 WHD is also required for the
OCCM to double hexamer transition. While exploring the Mcm7 WHD, we
discovered new rules and requirements for ATP hydrolysis dependent Cdt1
release. Finally, we identified novel mechanism in CDK dependent control of
helicase loading, which are important for genome stability. The work performed
as part of this thesis has resulted in the discovery of several new mechanisms,
which together provide fundamental insights into the function and regulation of
the highly complex eukaryotic helicase loading process.
factor during this process is the replicative helicase MCM2-7, a ring-shaped
hetero-hexameric protein assembly, which unwinds DNA at the replication fork.
In a multi-step process, ORC, Cdc6 and Cdt1 promote the loading of two
MCM2-7 hexamer into a head-to-head double hexamer around double stranded
DNA.
The recent cryo-EM structure of an ORC/Cdc6/Cdt1/MCM2-7 (OCCM)
intermediate has revealed that the MCM2-7 hexamer and ORC/Cdc6 are
connected by C-terminal winged helix domains (WHDs). However, how these
WHDs function for helicase loading and MCM2-7 double-hexamer formation
has been largely unknown. Using a combination of biochemical and structural
approaches, we have investigated the function of the different Mcm C-termini
during pre-RC formation.
In this study, we developed a new concept, explaining how distinct
functions of the individual WHDs drive the multi-step pre-RC formation process.
In particular, we discovered that the Mcm5 WHD serves as a key factor in order
to signal helicase loading defects and to induce quality control during pre-RC
formation. In addition, it was found that the Mcm5 WHD is also required for the
OCCM to double hexamer transition. While exploring the Mcm7 WHD, we
discovered new rules and requirements for ATP hydrolysis dependent Cdt1
release. Finally, we identified novel mechanism in CDK dependent control of
helicase loading, which are important for genome stability. The work performed
as part of this thesis has resulted in the discovery of several new mechanisms,
which together provide fundamental insights into the function and regulation of
the highly complex eukaryotic helicase loading process.
Version
Open Access
Date Issued
2018-09
Date Awarded
2018-12
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Speck, Christian
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)