Protein-protein interactions underlying damage checkpoint activation in S. pombe

Wardlaw, Christopher (2014) Protein-protein interactions underlying damage checkpoint activation in S. pombe. Doctoral thesis (PhD), University of Sussex.

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DNA damage can lead to the accumulation of mutations and diseases such as cancer. It
is therefore integral for cells to identify this damaged DNA and promote its repair. To
carry out this function eukaryotic cells have evolved signal transduction pathways
known as the DNA structure checkpoints. Much of the molecular mechanism
underlying these pathways is still far from understood. The work in this thesis uses the
model organism Schizosaccharomyces pombe to investigate these mechanisms, with a
particular focus on the TopBP1 homolog Rad4.
TopBP1 plays an essential scaffolding role in the initiation of DNA replication, but is
also a key protein in the DNA structure checkpoints. It has previously been shown in
metazoans and budding yeast to stimulate the kinase activity of ATR, via its ATR
Activation Domain (AAD), an early event in checkpoint activation. The work presented
in here, along with initial work carried by previous members of the Carr Laboratory;
Su-Jiun Lin and Valerie Garcia, shows that the Rad4TopBP1 AAD acts in a chromatin
dependent pathway to amplify the checkpoint signal in G1/S-phase, where DNA
resection is limited. A second AAD is also identified in the checkpoint clamp protein
Rad9, which acts redundantly with the Rad4 AAD.
As well as its AAD function, Rad4 also plays a scaffolding role in the DNA structure
checkpoint pathways. The work in this thesis, in collaboration with the Laurence Pearl
and Li Lin Du laboratories, identifies the molecular mechanism of the interaction
between Rad4 and the mediator protein Crb253BP1. It is shown that sequential
phosphorylation of Crb2 by Cdc2CDK is required for the interaction with BRCT domains
1 and 2 of Rad4 and checkpoint activation. It is also shown that Rad4 most likely does
not interact with Mrc1 or Slx4 in the S. pombe checkpoint pathways.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User: Library Cataloguing
Date Deposited: 25 Apr 2014 15:02
Last Modified: 18 Sep 2015 13:45

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