Genetic and biochemical analysis of the Hel308 Helicase in the Archaeon Haloferax volcanii

Lever, Rebecca (2020) Genetic and biochemical analysis of the Hel308 Helicase in the Archaeon Haloferax volcanii. PhD thesis, University of Nottingham.

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Abstract

Hel308 is a monomeric 3’ to 5’ DNA helicase conserved throughout archaea and metazoans, but is absent from bacteria and fungi. Hel308 acts analogously to RecQ-family helicases, which serve as guardians of genome stability. Hel308 has been implicated in a variety of overlapping DNA repair pathways, including homologous recombination. However, the exact role of Hel308 remains elusive. Haloferax volcanii was used as a model archaeon to study cellular role of Hel308.

Deletion mutants of hel308, or point mutants that affect the ATP-dependent helicase activity, are slow-growing and are sensitive to DNA cross-linking agents, which are potent blocks to DNA replication. By contrast, point mutants that exhibit hyper-recombinant phenotypes are not deficient in DNA repair or normal growth. These hyper-recombinant point mutations identify a separation of function of Hel308, where the ability to bind D-loop intermediates of recombination is affected, but the helicase activity involved in DNA repair is not.

Mutants deleted for radB, a recombination mediator, exhibit a decrease in recombination to approximately 5% of that in wild type cells. However, a double deletion of hel308 with radB restores, in part, homologous recombination. In this study, strains deleted for hel308 exhibited a 5-fold increase in recombination compared with wild-type, which suggests an anti-recombinase function for Hel308 and fits with its role as a guardian of genome stability.

Strains deleted for radA usually have a severe growth defect and are very sensitive to both UV irradiation and MMC treatment. Strains deleted for hel308 exhibit similar phenotypes, but not to the same severity as the ∆radA strains. Most interestingly, the strain deleted for both hel308 and radA generated in this study was healthier than either single deletion. These data suggest that in the absence of Hel308-mediated repair and homologous recombination, a third distinct DNA-repair pathway is activated, which is ordinarily supressed by both Hel308-mediated repair and homologous recombination. An alternative explanation is that in deleting both the hel308 and radA genes, a suppressor mutation has arisen. Only a singular clone with the ∆hel308∆radA phenotype was generated, which supports the suppressor-mutation theory. However, a suppressor mutation or mutations would need the ability to bypass two distinct repair pathways in order to restore wildtype-like fitness, which seems unlikely.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Allers, Thorsten
Bolt, Ed
Keywords: DNA helicase; Haloferax volcanii; Helicase activity; DNA replication; DNA repair
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: 60024
Depositing User: Lever, Rebecca
Date Deposited: 16 Aug 2021 07:44
Last Modified: 16 Aug 2021 07:44
URI: http://eprints.nottingham.ac.uk/id/eprint/60024

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