Investigating the role of Tyrosyl-DNA Phosphodiesterase 1 in nuclear and mitochondrial DNA repair

Chiang, Shih-Chieh (2017) Investigating the role of Tyrosyl-DNA Phosphodiesterase 1 in nuclear and mitochondrial DNA repair. Doctoral thesis (PhD), University of Sussex.

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Abstract

Damages to the genetic materials arise throughout the lifespan of a cell, and elicit
upregulation of DNA repair factors. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is part of
a DNA repair protein complex that specialises in the repair of DNA base modifications
and single-strand breaks (SSBs). TDP1 removes a broad spectrum of chemical
adducts from the 3’ end of a DNA strand break, including topoisomerase 1 (TOP1)
peptide, during DNA transcription and replication. Inactivation or deletion of TDP1 is
associated with cerebellar dysfunction and degeneration, with remarkably little extraneurological
manifestation. The reason for the selective dependence of the cerebellar
neurons on TDP1 activity is not clear. It was hypothesised that the TDP1 activity is
upregulated in tissues with high levels of SSBs, either from DNA transcriptional activity,
or reactive oxygen species (ROS)-induced damage.
The aim of this doctoral project was therefore to identify and characterise the cellular
mechanisms that regulate TDP1 activity. Our lab has previously shown that the Nterminus
domain (NTD) of TDP1 covalently interacts with DNA ligase 3α. In this thesis,
evidence has been presented to show that this interaction is regulated by the putative
ATM/ATR/DNA-PK phosphorylation site, serine 81, to prolong TDP1 half-life, and
enhance cellular survival after genotoxic stress. A second post-translational
modification in the NTD by SUMOylation of the K111 residue was identified,
enlightening a mechanism by which TDP1 is recruited to sites of transcription-mediated
SSBs.
To investigate the requirement for TDP1 in cells under high levels of oxidative stress, I
have developed a mouse cellular model whereby the levels of endogenous ROS can
be modulated by overexpression of the human anti-oxidant enzyme superoxide
dismutase 1 (SOD1) or its toxic mutant SOD1G93A. Overexpression of SOD1G93A in
Tdp1-/- MEFs induces accumulation of chromosomal SSBs and decreases survival after
H2O2 challenge, while overexpression of SOD1 has a protective effect. Besides repair
of ROS-induced TOP1-cc in the nucleus, TDP1 also repairs mitochondrial
topoisomerase 1-mediated DNA breaks. This role is required during transcription and
assembly of mitochondrial subunits of the electron transfer chain complexes, and has
direct impact on mitochondrial respiration and ROS production. Collectively, these data
provide mechanistic insights into regulation of TDP1-mediated chromosomal and
mitochondrial DNA repair.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QP Physiology > QP0501 Animal biochemistry > QP0551 Proteins, amino acids, etc. > QP0609.P53 Phosphodiesterase
Depositing User: Library Cataloguing
Date Deposited: 13 Jun 2017 11:17
Last Modified: 13 Jun 2017 11:17
URI: http://sro.sussex.ac.uk/id/eprint/68554

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