Characterization of non-homologus end joining in Mycobacterium tuberculosis using nanofluidics

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dc.contributor.authorPersson, Elin
dc.contributor.departmentChalmers tekniska högskola / Institutionen för life sciencessv
dc.contributor.departmentChalmers University of Technology / Department of Life Sciencesen
dc.contributor.examinerWesterlund, Fredrik
dc.contributor.supervisorBudida, Anusha
dc.contributor.supervisorPavlova, Evgeniya
dc.date.accessioned2023-06-20T08:16:29Z
dc.date.available2023-06-20T08:16:29Z
dc.date.issued2023
dc.date.submitted2023
dc.description.abstractDNA, 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.
dc.identifier.coursecodeBBTX60
dc.identifier.urihttp://hdl.handle.net/20.500.12380/306311
dc.setspec.uppsokLifeEarthScience
dc.subjectnon-homologous end joining
dc.subjectDNA repair
dc.subjectnanofluidics
dc.subjectMycobacterium
dc.subjecttuberculosis
dc.subjectKu
dc.subjectLigD
dc.subjectDNA-protein interactions
dc.titleCharacterization of non-homologus end joining in Mycobacterium tuberculosis using nanofluidics
dc.type.degreeExamensarbete för masterexamensv
dc.type.degreeMaster's Thesisen
dc.type.uppsokH
local.programmeBiotechnology (MPBIO), MSc
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