Structure/function analysis of the essential protein Rad4TopBP1 in S. pombe

Lin, Su-Jiun (2011) Structure/function analysis of the essential protein Rad4TopBP1 in S. pombe. Doctoral thesis (DPhil), University of Sussex.

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Rad4TopBP1, a multiple BRCT domain protein, is essential for initiation of replication and
participates in checkpoint responses following genotoxic treatment during the S and G2
phases of the cell cycle. Rad4 interacts with several proteins that are involved in
initiation of DNA replication (i.e. Sld2) or activation of Chk1 and/or Cds1 checkpoint
kinases (i.e. Rad9, Crb2, and Rad3). However, it remained unclear how Rad4 and its
homologues are regulated to coordinate these diverse functions. This PhD project
presents a comprehensive structure/function analysis of the fission yeast Rad4TopBP1.
In order to obtain separation of function mutants of Rad4, a hydroxylamine random
mutagenesis genetic screen was performed. However, we were not able to separate a
checkpoint activation function from replicative function. Rad4 being phosphorylated in
a Cdc2-dependent manner, the role of Rad4 phosphorylation by Cdc2 was investigated.
A mutant strain containing multiple mutations at Rad4 Cdc2 consensus phosphorylation
sites does not exhibit significant sensitivity to DNA damage or HU. In addition, Rad4
Cdc2 phosphorylation sites do not play a role in DNA re-replication. There is no
significant phenotypic effect observed after DNA damage in S. pombe strains expressing
a Rad4 protein deleted for a putative domain (RXL motif) interaction with cyclin, or
harboring mutations in putative sumoylation motifs, or C-terminus truncation.
In higher eukaryotes, TopBP1 binds and activates the ATR-ATRIP complex via an
ATR-activating domain (AAD) in order to activate a checkpoint function. We identified
a potential AAD in C-terminal of Rad4 in S. pombe. I show that Rad4 physically
associates with Rad3 in vitro in an AAD-dependent manner. S. pombe strains mutated in
the AAD show a slight sensitivity to DNA damage and HU. The rad4 AAD mutants do
not completely prevent Rad3-mediated G2/M checkpoint activation after DNA damage.
The sensitivity in a rad4 AAD mutant increases when damage occurs in S-phase, when
histone H2A phosphorylation is defective. I established an artificial checkpoint
induction system in the absence of exogenous lesions by targeting checkpoint proteins
onto chromatin in S. pombe. Interestingly, Rad4 AAD is essential for checkpoint
activation in this system. Because this checkpoint activation is independent of
ssDNA-RPA formation, the data suggest that the AAD plays a role in
chromatin-mediated checkpoint maintenance/amplification. Altogether, this pathway
seems to play an important role in S-phase when DSBs resection is limited.

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: 07 Mar 2011 10:41
Last Modified: 13 Aug 2015 15:06

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