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Abstract

DNA replication is fundamental to the proliferation of life. Sites of DNA replication initiation are called replication origins. Bacteria replicate from a single origin whereas eukaryotes utilise multiple origins for each chromosome. The archaeal domain includes species which replicate using multiple origins of replication in addition to those which use a single origin. Archaeal DNA replication proteins are similar to eukaryotic replication machinery. Most characterised archaeal origins are adjacent to an orc gene which encodes a homologue of the Orc1 subunit of the eukaryotic initiator protein complex.

Replication origins of the halophilic archaeon Haloferax volcanii were identified using a combination of genetic, biochemical and bioinformatic approaches. H. volcanii has a multireplicon genome consisting of a circular main chromosome and three mini-chromosomes: pHV1, pHV3 and pHV4. The major chromosome contains multiple origins of replication and is the first example of multiple origins on a single replicon in the Euryarchaeota. Each characterised origin is adjacent to an orc gene and contains repeated sequence motifs surrounding an A/T-rich duplex unwinding element.

The archaeal recombinase, RadA, is homologous to eukaryotic and bacterial Rad51/RecA. It is widely held that deletion of radA results in elimination of homologous recombination. In this study the discovery of a radA-independent recombination pathway specific to replication origins is described. This dynamic mechanism was identified by observing chromosomal integration of plasmids containing H. volcanii replication origins in a radA deletion strain.

The eukaryotic RAD25 gene is involved in nucleotide excision repair and transcription. H. volcanii has four RAD25 homologues, one on pHV4 and three near the oriC-2 locus on the main chromosome. A role for the assistance of oriC-2 firing is proposed based on autonomously replicating plasmid assays. Deletion of all four RAD25 homologues did not increase DNA damage sensitivity but resulted in a minor growth defect.