Abstract
BackgroundInformation transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential.ResultsUsing a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence). The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene.ConclusionThe results showed that ten out of nineteen eukaryotic-type DNA replication genes are essential for Halobacterium sp. NRC-1, consistent with their requirement for DNA replication. The essential genes code for two of ten Orc/Cdc6 proteins, two out of three DNA polymerases, the MCM helicase, two DNA primase subunits, the DNA polymerase sliding clamp, and the flap endonuclease.
Highlights
Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes
NRC-1, ten orc/cdc6 genes are present, with orc6, orc7, orc8, and orc10 genes located on the large chromosome, orc1-5 located on pNRC200, and orc9 located on both the pNRC100 and pNRC200 replicons
Phylogenetic reconstruction of Orc/Cdc6 protein sequences from sequenced haloarchaeal genomes and representative eukaryotes indicated the presence of five distinct haloarchaeal/archaeal clades, all distantly related to eukaryotic Orc1 and Cdc6 (Fig. 2)
Summary
Bioinformatic analysis of Orc/Cdc Halophiles are unique among the Archaea in possessing a large gene family of Orc/Cdc genes, as other archaeal organisms most commonly encode only two Orc/Cdc homologs [8]. After isolation and screening of 40 Foar colonies via PCR, we found that deletion alleles could not be recovered for either gene of the D family DNA polymerase, polD1 and polD2, or for the gene encoding the chromosomally encoded B family polymerase, polB1, indicating that they are essential to this organism (Fig. 4A and Table 2). Individual colony isolates were screened for the presence of wild-type or deletion alleles of the chromosomal copy of the gene of interest, in the same manner that the aforementioned non-essential gene knockout strains were screened (Figs 4B, 5B, 6B and Table 2). Our results showed that one or more chromosomal deletants were obtained for polD1, polB1, mcm, pri, and rad genes (Figs 4B, 5B, 6B, and Table 2) only when a complementing wild-type copy was provided on a replicating plasmid These results confirm the requirement of the five genes for cell viability using both statistical and genetic criteria. N.A – Not Applicable exogenous selection, unequivocally displaying the essential nature of the DNA replication gene carried on the plasmid (data not shown)
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