Role of N-terminal domains of p400 ATPase in the ATM interaction and DNA damage response : a thesis presented in partial fulfillment of the requirements for a the degree of Master of Science (MSc) in Genetics at Massey University, Manawatū, New Zealand
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Date
2016
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Massey University
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Abstract
Efficient repair of damaged DNA and preservation of genomic integrity is
integral in the maintenance of proper cellular function and prevention of unrestricted
cell proliferation. One critical threat to the stability of the genome is the double strand
break (DSB), arguably one of the most cytotoxic lesions to DNA. Interference with the
DSB repair mechanism can lead to dysregulation of cellular systems and the prospective
development of malignancies. Two critical proteins in DBS repair are the Ataxia
Telangiectasia Mutated (ATM) kinase, a serine/threonine kinase from the
Phosphatidylinositol 3-Kinase-related Kinase (PIKK) family, and p400, an ATPase
chromatin remodeler. ATM is one of the first responders to DSBs and is responsible for
the phosphorylation of a multitude of protein substrates including the histone variant
H2AX. Beyond its phosphorylation ability, ATM has been proposed as a potential
shuttle for other repair machinery, aiding in the early and efficient recruitment of
proteins to the DNA damage foci. One such proposed protein is p400. The exact role of
p400 in DSB repair is unknown but previous studies show that there is a decrease in
repair efficiency in its absence. A prospective interaction is supported by previous
studies in which p400 and p400 N-terminal derivatives co-immunoprecipitate with
ATM in vivo in HEK293T cells.
This study aimed to confirm the interaction of ATM and p400 N-terminal
derivatives in vitro and explore the functional implications of the association in vivo in
U2OS cells. It was not possible to isolate full-length p400 derivatives in vitro and thus
no conclusive results were obtained. Functional assays revealed the ability of one p400
fragment, F1, to inhibit DNA repair and cell proliferation after DNA double-strand
break induction with bleomycin. Ectopic expression of the other two p400 N-terminal
fragments, F2 and F3, induced an inhibition of cell proliferation under standard growth
conditions. Although no conclusive results were acquired, a trend emerged suggesting
that N-terminal fragment F1 is able to interfere with ATM protein-protein interactions
resulting in a decrease in the efficiency of the DNA damage response and repair. These
results implicate F1 as a potential target for further research in both DNA repair and
cancer therapy.
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Keywords
DNA repair, Cancer cells, Growth, Regulation, Protein kinases, Inhibitors, Therapeutic use, Chromatin, Structure, Cancer, Gene therapy, Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::Genetics