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Title: Dissecting the DNA damage response : structural and biochemical insights into the Mec1-Ddc2 checkpoint kinase complex
Author: Sawicka, Marta
ISNI:       0000 0004 6348 3273
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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The survival and proper functioning of an organism depends on the faithful replication of its genetic material. The yeast checkpoint kinase Mec1 and its integral partner Ddc2 (human ATR and ATRIP respectively) play a pivotal role in initiating checkpoint signalling as a response to DNA damage. Mec1 is activated by cell cycle speci c activators that act through conserved aromatic residues located in their unstructured C-terminal regions. Since the molecular details of the activation mechanism of Mec1 are not fully understood yet, we studied the structure of the Mec1-Ddc2 complex and its interactions with the activator Dpb11 protein. Our ndings provide the rst insights into the overall architecture of Mec1-Ddc2 and serve as a basis for a proposed framework for the activation of Mec1. Our single-particle cryo-electron microscopy reconstruction shows Mec1- Ddc2 exists as a dimer of heterodimers and associates through intertwining arm regions consisting of the N-terminal Mec1 HEAT repeats of the adjacent monomers. The Ddc2 subunit, which extends from these repeats, further stabilises the oligomerization interface. The head of the structure accommodates the kinase domain located at the conserved C-terminus of Mec1. Due to a head-to-head dimer conformation, the kinase domains face each other although they are fully separated indicating they do not cause structural impediments that would block substrate access. The interactions between the N-terminus of Ddc2 and the C-terminal tail of Dpb11 mediate the recruitment of the activator to the checkpoint complex. The Ddc2 subunit, which neighbours the kinase domain, not only provides structural support but also facilitates Mec1 activation by bringing Dpb11 into close proximity to the active site. We propose a model where the activator stimulates the kinase activity via multiple interactions between the Mec1 and Ddc2 subunits of the complex that trigger small allosteric changes within the kinase domains. We hope such insights will pave the way to a full mechanistic understanding of this important signalling pathway.
Supervisor: Zhang, Xiaodong Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral