Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in E. coli cell physiology

Reeves, Ryan Peter (2019) Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in E. coli cell physiology. MRes thesis, University of Nottingham.

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Abstract

CRISPR-Cas is an adaptive immune system which provides protection from invasive mobile genetic elements in prokaryotes. CRISPR-Cas immunity starts with acquisition: this involves the capture of DNA fragments called ‘prespacers’, which are then processed to become ‘protospacers’ and integrated into the host’s chromosome as ‘spacers’. Acquisition is catalysed by the Cas1-2 complex along with host factors. Although the spacer integration mechanism is well understood, the prespacer capture process remains cryptic. In the E. coli CRISPR I-E system, the products of replication fork collisions have been linked to prespacer capture by Cas1-2. By increasing the frequency of replication-fork collisions using an E. coli strain with an additional ectopic origin of replication, I show that the level of spacer acquisition increases in vivo compared to wild-type; providing evidence that RecBCD-helicase processed fragments from stalled DNA replication forks are captured as prespacers by Cas1-2. Furthermore, I demonstrate that spacer acquisition increases in E. coli under nutritional stress. I also show that N-terminal tags on Cas1 have a detrimental effect on acquisition in vivo, despite successful spacer integration activity in vitro; as well as investigating the role of Cas3 in a cell stress response; and initiating genetic work into the unusual CRISPR-Cas system of Marinitoga piezophila.

Item Type: Thesis (University of Nottingham only) (MRes)
Supervisors: Bolt, Ed
Chalmers, Ronald
Keywords: CRISPR-Cas immunity; Prespacer capture process; Spacer acquisition; Cell stress response
Subjects: Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 56480
Depositing User: Reeves, Ryan
Date Deposited: 19 Jul 2019 04:40
Last Modified: 07 May 2020 11:45
URI: http://eprints.nottingham.ac.uk/id/eprint/56480

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