Serdyuk, Anastasia
(2024)
Origins “R” us: investigating the role of R-loops in origin-independent DNA replication.
MRes thesis, University of Nottingham.
Abstract
The ability of a molecule to self-replicate has been implicated to be the driving force behind the evolution of cellular life from the primordial RNA world. Thus the physicochemical properties of genomic replication are conserved in all three domains of life; Eukaryotes, Bacteria, and Archaea. One of these fundamental properties is the requirement of a terminal hydroxyl group for de novo DNA synthesis. The canonical DNA replication mechanism involves initiation from specific chromosomal sequences – origins of replication. However, an alternative mechanism – recombination dependent replication – has been observed in every domain; the cells are able to replicate without an origin while utilising the 3’ end of a recombination intermediate (directly from R-loops, or indirectly from D-loops) to initiate synthesis from any location on the chromosome. Our understanding of the steps and enzymology of the full replisome assembly from recombination intermediates remains fragmentary. This is due to the small number of culturable model organisms that can replicate in an origin-independent manner. One of these organisms is the halophilic archaeon Haloferax volcanii, which growths faster in the absence of origins, but is easy to culture and is amenable to genetic manipulation. H.volcanii possesses a unique genome architecture: a main chromosome, and 3 mini-chromosomes; each containing multiple origins which can all be deleted except for the origin on pHV3. Moreover, attempted deletions of the pHV3 origin have resulted in genomic rearrangements, where pHV3 is integrated onto the main chromosome. The reasons for that are unknown, except for the low transcription levels on pHV3 detected in previous transcriptomic analyses.
To investigate the correlation between the levels of transcription, and the ability to delete the origin on pHV3, we have generated strains with increased transcription levels on pHV3 by employing three parallel research lines: through (1) engineering a tryptophan-inducible promoter for regulatory expression of the adh2 gene on the pHV3 mini- chromosome, and (2) generating deletion constructs of rnhA and rnhC genes which degrade R-loops. Another way is (3) to stabilise the D-loop intermediate structure by attempting to delete the origin by transforming Hel308 deletion (∆Hel308) and point mutation (Hel308-D145N, Hel308-F316A, Hel308- R743A) strains with plasmids carrying ∆ori-pHV3, ∆orc6 (initiator protein gene), and ∆ori-pHV3 with simultaneous deletion of orc6.
Our results – for the first time – confirm the possibility of origin deletion on pHV3 in wild-type and Hel308-R743A, strains, orc6 initiator deletion (∆orc6) in Hel308-D145N, Hel308-F316A, and Hel308-R743A point mutation strains, as well as combined pHV3 origin with orc6 deletions in ∆Hel308 and Hel308-R743A strains. This supports the initial hypothesis that the deletion of Hel308 may be involved in the stabilisation of D-loop structures, and be a key factor in the replisome assembly process.
However, it remains an open question whether this origin-deletion has occurred with pHV3 maintaining its episomal state. For that, further analysis using pulsed-field gel electrophoresis would confirm if pHV3 had integrated onto the main chromosome.
Overall, our work provides the foundational basis to warrant further study in the correlation between the increased levels of available recombination intermediates – through the stabilisation of D-loops/R-loops, or upregulation of gene expression, and hence transcription – and the deletion of origins in the model archaeon Haloferax volcanii.
Key Words: DNA replication; DNA Recombination; Recombination-Dependent Replication; D-loops; R-loops; Hel308; Archaea; Halophiles; Haloferax volcanii; Genome Architecture; Origin Deletion; Origins of Life; Evolution
| Item Type: |
Thesis (University of Nottingham only)
(MRes)
|
| Supervisors: |
Allers, Thorsten
Bolt, Ed
Loose, Matthew |
| Keywords: |
DNA replication; DNA Recombination; Recombination-Dependent Replication; D-loops; R-loops; Hel308; Archaea; Halophiles; Haloferax volcanii; Genome Architecture; Origin Deletion; Origins of Life; Evolution |
| Subjects: |
Q Science > QH Natural history. Biology > QH426 Genetics |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
78524 |
| Depositing User: |
Serdyuk, Anastasia
|
| Date Deposited: |
16 Jul 2024 04:40 |
| Last Modified: |
16 Jul 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/78524 |
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