Abstract
The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle.
Highlights
In all prokaryotic organisms, and in certain unicellular eukaryotes, DNA replication is thought to initiate at welldefined chromosomal sites
Archaea have circular chromosomes like bacteria but use enzymes similar to those found in eukaryotes to replicate their DNA
Few archaeal species have systems for genetics, and this has limited our understanding of DNA replication
Summary
In certain unicellular eukaryotes, DNA replication is thought to initiate at welldefined chromosomal sites. Our genetic observations indicate that while the functions of initiator proteins at least partially overlap in H. volcanii, the replication origins oriC1 and ori-pHV1/4 play a key role in the replication of their corresponding chromosomes. This suggests that duplication of replication origins, and not of the initiator genes, could have allowed the H. volcanii genome to develop toward a multireplicon structure. This process might still be continuing, since there are important differences between our data and the assembled genome sequence. The H. volcanii DS2 strain was used to isolate DNA for sequencing (Table 3), whereas genetic and biochemical experiments used derivatives of H. volcanii WFD11 or DS70 [19,20]
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