Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements
DNA replication typically initiates at origins, DNA sequences at which replication initiator proteins bind and instigate formation of the DNA replication machinery. It was thought that origins of replication are essential across all domains of life. However, the halophilic archaeon Haloferax volcani...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2023
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| Online Access: | https://eprints.nottingham.ac.uk/72357/ |
| _version_ | 1848800735664275456 |
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| author | Hodson, Katie |
| author_facet | Hodson, Katie |
| author_sort | Hodson, Katie |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | DNA replication typically initiates at origins, DNA sequences at which replication initiator proteins bind and instigate formation of the DNA replication machinery. It was thought that origins of replication are essential across all domains of life. However, the halophilic archaeon Haloferax volcanii can survive in the absence of origins, and in fact, strains lacking origins grow more quickly than the wild-type strain. When all origins are deleted, the archaeal RecA family recombinase RadA becomes essential. It is for this reason that it is thought that in the absence of origins, H. volcanii employs recombination-dependent replication.
In this study, Chromatin Immunoprecipitation Sequencing (ChIP-Seq) was undertaken using FLAG-tagged RadA, in a strain in which all origins were present on the main chromosome. This revealed a relative lack of RadA binding upon mini chromosome pHV3, extensive binding across mini chromosome pHV1, and a greater proportion of peaks mapping to promotor-transcription start site regions than to transcription termination sites or within exons. These findings demonstrate a tentative link between transcription and recombination; however, aspects of the protocol require optimisation.
Prior to this study, H. volcanii strains were generated in which the level of transcription of the mini chromosome pHV3 was dramatically increased; it was then found that the origin of replication on pHV3 may be deleted. This work established that in these strains, pHV3 had integrated onto the main chromosome – a phenomenon not previously seen in attempts to delete the pHV3 origin. This suggests that significant changes in transcription may lead to genome rearrangements. Oxford Nanopore MinION sequencing showed that integration of pHV3 on the main chromosome most likely occurred via recombination between two homologous ISH18 elements (transposase genes), one on pHV3 and the other on the main chromosome. Similar recombination events leading to large scale rearrangements of the H. volcanii genome architecture have been noted previously.
Given that ChIP-Seq revealed a lack of RadA binding to wilt-type pHV3, it could be hypothesised that the significant increase in transcription of pHV3 had led to an increase in RadA binding, thereby facilitating recombination of pHV3 with the main chromosome.
Overall, this study shows the interplay between recombination, transcription, and origin-independent replication. This study also demonstrates the efficacy of Oxford Nanopore MinION sequencing for H. volcanii strain verification. |
| first_indexed | 2025-11-14T20:56:17Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-72357 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:56:17Z |
| publishDate | 2023 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-723572023-07-31T04:40:30Z https://eprints.nottingham.ac.uk/72357/ Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements Hodson, Katie DNA replication typically initiates at origins, DNA sequences at which replication initiator proteins bind and instigate formation of the DNA replication machinery. It was thought that origins of replication are essential across all domains of life. However, the halophilic archaeon Haloferax volcanii can survive in the absence of origins, and in fact, strains lacking origins grow more quickly than the wild-type strain. When all origins are deleted, the archaeal RecA family recombinase RadA becomes essential. It is for this reason that it is thought that in the absence of origins, H. volcanii employs recombination-dependent replication. In this study, Chromatin Immunoprecipitation Sequencing (ChIP-Seq) was undertaken using FLAG-tagged RadA, in a strain in which all origins were present on the main chromosome. This revealed a relative lack of RadA binding upon mini chromosome pHV3, extensive binding across mini chromosome pHV1, and a greater proportion of peaks mapping to promotor-transcription start site regions than to transcription termination sites or within exons. These findings demonstrate a tentative link between transcription and recombination; however, aspects of the protocol require optimisation. Prior to this study, H. volcanii strains were generated in which the level of transcription of the mini chromosome pHV3 was dramatically increased; it was then found that the origin of replication on pHV3 may be deleted. This work established that in these strains, pHV3 had integrated onto the main chromosome – a phenomenon not previously seen in attempts to delete the pHV3 origin. This suggests that significant changes in transcription may lead to genome rearrangements. Oxford Nanopore MinION sequencing showed that integration of pHV3 on the main chromosome most likely occurred via recombination between two homologous ISH18 elements (transposase genes), one on pHV3 and the other on the main chromosome. Similar recombination events leading to large scale rearrangements of the H. volcanii genome architecture have been noted previously. Given that ChIP-Seq revealed a lack of RadA binding to wilt-type pHV3, it could be hypothesised that the significant increase in transcription of pHV3 had led to an increase in RadA binding, thereby facilitating recombination of pHV3 with the main chromosome. Overall, this study shows the interplay between recombination, transcription, and origin-independent replication. This study also demonstrates the efficacy of Oxford Nanopore MinION sequencing for H. volcanii strain verification. 2023-07-31 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/72357/1/Corrected_thesis_14322881_Katie_Hodson_MRes.pdf Hodson, Katie (2023) Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements. MRes thesis, University of Nottingham. Chromatin Immunoprecipitation Sequencing; FLAG-tagged RadA; RadA binding; Origin-independent replication; Recombination-dependent replication |
| spellingShingle | Chromatin Immunoprecipitation Sequencing; FLAG-tagged RadA; RadA binding; Origin-independent replication; Recombination-dependent replication Hodson, Katie Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements |
| title | Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements |
| title_full | Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements |
| title_fullStr | Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements |
| title_full_unstemmed | Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements |
| title_short | Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements |
| title_sort | origin-independent replication in haloferax volcanii: rada localisation, r-loops, and genomic rearrangements |
| topic | Chromatin Immunoprecipitation Sequencing; FLAG-tagged RadA; RadA binding; Origin-independent replication; Recombination-dependent replication |
| url | https://eprints.nottingham.ac.uk/72357/ |