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Walker, Emily (2019) Investigation of CRISPR-Cas adaptation mechanism through exploration of Cas4-1 fusions. PhD thesis, University of Nottingham.

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Abstract

CRISPR-Cas is an adaptive immune system present in bacteria and archaea. It involves two linked stages: adaptation and interference. Adaptation generates a database of mobile genetic element sequences within a CRISPR locus through ‘capture’ of DNA fragments by Cas1-Cas2 and integration into the CRISPR locus. The CRISPR locus is transcribed and separated into individual units, called crRNA. During interference crRNA bound within ‘interference’ ribonuceloprotein complexes targets DNA from Mobile genetic elements (MGE) through complementary base pairing between crRNA and the MGE before degradation of the target.

Adaptation is catalysed by Cas1 and Cas2 proteins in three contexts: naïve, targeted and primed adaptation. The pathway taken is dependent on whether the organism has encountered the MGE previously. A crucial stage of adaptation is the generation of fragments for ‘capture’, however the mechanism is unknown. In bacteria several proteins have been hypothesised to be involved in supporting adaptation including RecB, RecG, PriA and Cas4.



Cas4-1 fusions are a naturally occurring fusion of Cas4 and Cas1. Cas4-1 proteins from Methanosaeta harundinacea and Pyrinomonas methylaliphatogenes were selected for investigating the role of Cas4-1 in adaptation and to establish a single in vitro reaction for naïve adaptation. M. harundinacea Cas4-1 did not produce tractable protein, however a high yield of active Cas4-1 was obtained from P. methylaliphatogenes along with potential interacting partners (Cas2 and HPS).Cas4-1 was shown to contain active sites from both Cas1 and Cas4 through sequence alignment and in vitro biochemistry demonstrated Cas4-1 existed as a dimer in solution. Cas4-1 bound DNA with ssDNA regions and cleavage both ssDNA and dsDNA. No physical interaction was observed between Cas4-1 and Cas2 or HPS, but Cas2 was shown to be activator of Cas4-1 nuclease activity. This work demonstrates the activities of Cas4-1 and proposes a role for the protein in adaptation.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Bolt, Edward
Keywords:	CRISPR adaptation; Cas4-1
Subjects:	Q Science > QP Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	56071
Depositing User:	Walker, Emily
Date Deposited:	19 Jul 2019 04:40
Last Modified:	19 Jul 2021 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/56071

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