Characterization of non-homologus end joining in Mycobacterium tuberculosis using nanofluidics
Typ
Examensarbete för masterexamen
Master's Thesis
Master's Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2023
Författare
Persson, Elin
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
DNA, the building block of all life, is constantly exposed to various pressures that
can damage this vital molecule. These damages, if not repaired correctly, can result
in mutations, disease and possibly cell death; why the cell has developed several
ways to defend itself from the inevitable occurrence of DNA damages. The most
dangerous form of DNA damage is double stranded breaks, for which the cell has
only two repair mechanisms. One of these, non-homologous end joining, is especially
important due to the fact that it repairs these dangerous breaks in situations where
no template is available. However, the bacterial non-homologous end joining system
lacks comprehensive understanding, especially on the level of individual molecules.
Hence, the primary objective of this thesis is to explore the mechanisms of the
two key players of this system. While the human system is a complex interplay
of numerous proteins, the bacterial system primarily consists of two proteins that
are homologous to their eukaryotic counterparts: the homodimer Ku and DNA
ligase D. This project aimed to express, purify and utilize these two proteins from
Mycobacterium tuberculosis to investigate their interaction with DNA in both bulk
phase and with single molecule assays, especially a nanofluidic device based on
confinement of DNA. Our findings reveal that the C-terminal arm of the Ku protein,
as well as the nature of the double-stranded break, plays a critical role of DNA
binding and bridging. The Ku from Mycobacterium tuberculosis was also found to
have the ability to bridge blunt ended DNA, a characteristic not observed in other
bacterial species such as Bacillus subtilis. Further, it was proven by single molecule
methods that the ligation of double stranded breaks is directly dependent on efficient
bridging of the DNA by the Ku homodimer.
Beskrivning
Ämne/nyckelord
non-homologous end joining , DNA repair , nanofluidics , Mycobacterium , tuberculosis , Ku , LigD , DNA-protein interactions