Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements

Hodson, Katie (2023) Origin-independent replication in Haloferax volcanii: RadA localisation, R-loops, and genomic rearrangements. MRes thesis, University of Nottingham.

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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.

Item Type: Thesis (University of Nottingham only) (MRes)
Supervisors: Allers, Thorsten
Dattani, Ambika
Gray, Stephen
Keywords: Chromatin Immunoprecipitation Sequencing; FLAG-tagged RadA; RadA binding; Origin-independent replication; Recombination-dependent replication
Subjects: Q Science > QR Microbiology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 72357
Depositing User: Hodson, Katie
Date Deposited: 31 Jul 2023 04:40
Last Modified: 31 Jul 2023 04:40
URI: https://eprints.nottingham.ac.uk/id/eprint/72357

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