Smith, Victoria Emily
(2022)
Atypical DNA replication in Haloferax volcanii in the absence of replication origins.
PhD thesis, University of Nottingham.
Abstract
Replication origins are the sites of initiation of DNA replication. Origins are universal and have been assumed to be essential. However, the halophilic archaeon Haloferax volcanii is able to survive in the absence of origins, in fact growing faster than its wild-type counterpart. Replication in the absence of origins has been proposed to depend on replication-dependent replication (RDR), due to the requirement in the origin-deleted H. volcanii mutant for the recombinase protein, RadA.
In the work presented here, treatment of origin-deleted H. volcanii mutants revealed a tolerance of the PolB-specific inhibitor, aphidicolin, which increased with each origin deletion. While this suggests that originless strains may have a reduced requirement for the Family B DNA polymerase PolB, the replicative polymerases, PolB and PolD were found to be essential in both origin-containing and origin-deleted strains.
During eukaryotic DNA replication, the Cdc45 protein forms the CMG replicative helicase complex, in conjunction with MCM helicase and GINS. Archaeal species, including H. volcanii, encode counterparts of the MCM and GINS components of the CMG complex. Studies in Thermococcus kodakarensis have revealed that the GINS-associated nuclease (GAN), a RecJ- family protein, adopts the role of Cdc45. H. volcanii encodes four RecJ proteins (RecJ1-4). Phylogenetic, genetic and biochemical analysis carried out here suggests that RecJ1 acts as GAN but the role of RecJ2 remains unknown, while RecJ3 and RecJ4 have roles alongside the DNA repair protein Hef. All recJ genes were found to be dispensable in H. volcanii, with the exception of recJ2; overexpression of RecJ2 from an ectopic site was not sufficient to compensate for recJ2 deletion from the wild type locus. Phenotypic analysis of recJ mutants in H. volcanii has shed light on the possible functions of these proteins, however questions remain around their specific roles.
Previous data has shown an increased requirement for essential replicative helicase MCM in the absence of DNA replication origins. GINS is essential in eukaryotes and the same is assumed for archaea. It was determined here that deletion of GINS is not possible in H. volcanii, but strains with inducible ginS alleles suggested that the requirement for GINS does not match that of its fellow CMG complex member MCM.
The interplay between DNA polymerases, replication origins and the CMG complex warrants further work. Differential usage of polymerases and CMG proteins in the presence or absence of origins could provide critical information on the mechanisms of both canonical and recombination- dependent DNA replication in archaea.
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