Harrison, Catherine
(2024)
Role of Hel308 helicase in origin-independent DNA replication in two species of Haloferax.
PhD thesis, University of Nottingham.
Abstract
DNA replication is a process which is essential to all forms of life. This process is initiated at defined points, known as origins of replication. Origins are universal, and were previously believed to be essential. However, the halophilic euryarchaeon Haloferax volcanii is able to survive in the absence of origins, and in fact, origin-deleted mutants grow faster than the wild-type. Growth in these circumstances is thought to be dependent on recombination-dependent replication, since the recombinase protein RadA is essential in origin-deleted cells.
The Superfamily 2 DNA helicase Hel308 plays a similar role to RecQ family helicases, and has been implicated in genome stability and DNA repair. Like RecQ, Hel308 has a preference for ssDNA and forked structures in vitro. The hel308 gene is inessential in H. volcanii, and its deletion is associated with growth defects and increased susceptibility to DNA damage. ATPase-null point mutants of this protein have been explored previously, with one (K53A) reported to be lethal in both H. volcanii and E coli.
In the closely related species Haloferax mediterranei, origins have been shown to be essential, in contrast to H. volcanii. It was also previously reported that the hel308 gene from this species was essential. A genetic screen previously identified this gene as a potential inhibitor of origin-independent replication in H. volcanii.
In the work presented here, hel308 and its relevance to origin-independent replication were explored. It was found that H. mediterranei hel308 does not prevent origin-independent replication in H. volcanii, either through a system with an inducible origin, or through inducible expression of hel308 in a strain lacking origins.
The ATPase-null point mutant was not found to be lethal in H. volcanii, and could be engineered onto the chromosome. Brief exploration of this strain revealed a phenotype similar to both Δhel308 and previous ATPase-null point mutants, but not exactly equivalent.
Attempts were made to identify the factors contributing to the essentiality of hel308 in H. mediterranei, which questioned the original report of essentiality of this gene. Despite further investigation, essentially of hel308 in H. mediterranei has not been conclusively proven or disproven.
Origin-deleted cells also show increased resistance to the family B DNA polymerase inhibitor aphidicolin, suggesting a differential usage of DNA polymerases in these cells. Meanwhile, hel308-deleted cells show increased susceptibility to aphidicolin. Responses to aphidicolin were explored in H. volcanii strains with and without origins, and with various hel308 alleles present or absent. The susceptibility to aphidicolin conferred by hel308 deletion appears dominant to the aphidicolin resistance seen in originless strains. However, very low doses of aphidicolin induced a small increase in growth rate in strains lacking origins and with functional Hel308 present. It is suggested that these effects may be due to differential DNA polymerase usage and the importance of Hel308 in processing stalled forks following aphidicolin treatment. Preliminary results concerning the response of H. mediterranei to aphidicolin are intriguing, but do not shed conclusive light on its workings.
The interplay between origins, recombination and repair are complex, and warrant further work. Differential usage of DNA polymerases in the presence or absence of origins could provide important insight into the mechanisms of origin-initiated and recombination-dependent replication in archaea.
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