Investigating the role of 70 bp repeats and VSGs in formation of DNA double strand breaks in Trypanosoma brucei bloodstream expression sites

Salthouse, Holly Rebecca (2025) Investigating the role of 70 bp repeats and VSGs in formation of DNA double strand breaks in Trypanosoma brucei bloodstream expression sites. MSc(R) thesis, University of Glasgow.

Abstract

Trypanosoma brucei are protozoan parasites, which evade the immune system by antigenic variation with the use of a variant surface glycoprotein (VSG). A double strand DNA break (DSB) has been proposed as the first step in VSG switching and it has previously been demonstrated that when DNA breaks arise in the active expression site, this leads to accumulation of RNA-DNA hybrids, R-loops, however, there is still uncertainty of how and where the double stranded breaks form. Mapping of DNA breaks in the expression site by BLISS has implicated two key features in the formation of the breaks: the VSG 3’ end where the break is found and in the 70 bp repeats. This project aimed to examine the relative contribution of both the VSG 3’ end and the 70 bp repeats to the formation of a DNA break and VSG switching. To address this, bloodstream form T. brucei cell lines with alterations in the active bloodstream expression site (BES) were generated using CRISPR/Cas9. Two regions of 70 bp repeats were successfully removed and a distinct VSG sequence (VSG121) was relocated to two other locations within the active BES. In addition, a plasmid containing the VSG of the active expression site in this cell line (VSG221) was successfully generated. To confirm alterations to the active BES PCR screening, VSG immunofluorescence, and Oxford Nanopore sequencing were performed. It was identified by Oxford Nanopore sequencing that when the large section of 70 bp repeats was removed, the smaller section of 70 bp repeats expanded. In future work, these cell lines will allow for investigation of R-loops and double strand breaks by comparison of DRIP-seq and INDUCE-seq/BLISS data with the natural organisation of the active BES and so, give an insight into whether the 70 bp repeats and/or VSG sequence drive R-loop and DNA DSB formation and if they are necessary for break formation linked to VSG switching.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Subjects: Q Science > QR Microbiology; Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Supervisor's Name: McCulloch, Professor Richard
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-84899
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 Feb 2025 10:19
• Last Modified: 17 Feb 2025 10:20
• Thesis DOI: 10.5525/gla.thesis.84899
• URI: https://theses.gla.ac.uk/id/eprint/84899