Publication Date

2008

Document Type

Dissertation

Committee Members

Steven Berberich (Committee Member), David Cool (Committee Member), Patrick Dennis (Committee Member), James Mcdougal (Committee Chair), John Turchi (Advisor)

Degree Name

Doctor of Philosophy (PhD)

Abstract

Ionizing radiation (IR) and radiomimetic drugs used in cancer chemotherapy cause DNA double-strand breaks which are repaired by the nonhomologous end joining (NHEJ) pathway. Ku is a heterodimeric protein comprised of 70 and 80 kDa subunits and recognizes free DNA ends. Once Ku is bound to DNA, it binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and forms a heterotrimeric DNA-PK complex for repair and damage signaling.

We have analyzed the Ku protein using multiple biochemical techniques and uncovered a novel reversible redox change (Andrews, Lehman and Turchi, (2006) JBC 281(19):13596-603). From this data, we hypothesized that a redox-dependent conformational change was responsible for the unexplained differences in our DNA binding assays. This was confirmed by limited proteolysis and mass spectrometry analysis of oxidized and reduced Ku. The conformational change was manifested by alterations in the Ku70 subunit in proximity to the DNA binding site. Our results suggest a model for a Ku-DNA interaction that is altered by redox status upon DNA damage in the cell.

In order to understand the complex structure of DNA-PK, we have initially examined Ku-DNA interactions using advanced biochemical techniques. Structural studies on Ku by other groups have utilized truncated versions missing disordered C-terminal domains (CTDs) from both subunits. Chemical modification with NHS-biotin and mass spectrometry were used to identify biotinylated reactive lysines. Biotinylation of free Ku revealed several lysines on Ku which were reduced or eliminated upon DNA binding. Interestingly, in the predicted C-terminal SAP domain of Ku70, biotinylation patterns suggest a structural change at this site induced by DNA binding. Limited proteolytic digests of free and DNA-bound Ku revealed a series of unique peptides correlating to a change in the accessibility of the Ku70 and Ku80 CTDs. A 10 kDa peptide was also identified which was preferentially generated under non-DNA-bound conditions and mapped to the Ku70 CTD. These results indicate a DNA-dependent movement or structural change in the CTDs of Ku70 and Ku80 that may contribute to DNA-PKcs binding and activation. This is the first demonstration of DNA-dependent conformational changes in Ku and will aid in discerning the mechanism of DNA-PK activation.

Page Count

200

Department or Program

Biomedical Sciences

Year Degree Awarded

2008

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