Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in E. coli cell physiology
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 t...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2019
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| Online Access: | https://eprints.nottingham.ac.uk/56480/ |
| _version_ | 1848799335624474624 |
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| author | Reeves, Ryan Peter |
| author_facet | Reeves, Ryan Peter |
| author_sort | Reeves, Ryan Peter |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T20:34:02Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-56480 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:34:02Z |
| publishDate | 2019 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-564802025-02-28T14:28:45Z https://eprints.nottingham.ac.uk/56480/ Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in E. coli cell physiology Reeves, Ryan Peter 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. 2019-07-19 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en arr https://eprints.nottingham.ac.uk/56480/1/Ryan%20Reeves%20MRes%20Thesis%204312537%20Corrected.pdf Reeves, Ryan Peter (2019) Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in E. coli cell physiology. MRes thesis, University of Nottingham. CRISPR-Cas immunity; Prespacer capture process; Spacer acquisition; Cell stress response |
| spellingShingle | CRISPR-Cas immunity; Prespacer capture process; Spacer acquisition; Cell stress response Reeves, Ryan Peter Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in E. coli cell physiology |
| title | Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in
E. coli cell physiology |
| title_full | Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in
E. coli cell physiology |
| title_fullStr | Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in
E. coli cell physiology |
| title_full_unstemmed | Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in
E. coli cell physiology |
| title_short | Analysis of Cas1-2 and Cas3 CRISPR immunity proteins in
E. coli cell physiology |
| title_sort | analysis of cas1-2 and cas3 crispr immunity proteins in
e. coli cell physiology |
| topic | CRISPR-Cas immunity; Prespacer capture process; Spacer acquisition; Cell stress response |
| url | https://eprints.nottingham.ac.uk/56480/ |