| Summary: | <p>Abstract</p> <p>Background</p> <p>Information 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, <it>Halobacterium </it>sp. NRC-1, and used it to determine which DNA replication genes are essential.</p> <p>Results</p> <p>Using a directed in-frame gene knockout method in <it>Halobacterium </it>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 <it>orc</it>/<it>cdc6 </it>genes in <it>Halobacterium </it>sp. NRC-1, only two were found to be essential, <it>orc10</it>, on the large chromosome, and <it>orc2</it>, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, <it>polB1</it>, and the chromosomally encoded euryarchaeal-specific D family, <it>polD1/D2 </it>(formerly called <it>polA1/polA2 </it>in the <it>Halobacterium </it>sp. NRC-1 genome sequence). The pNRC200-encoded B family polymerase, <it>polB2</it>, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, <it>mcm</it>, the eukaryotic-type DNA primase, <it>pri1/pri2</it>, the DNA polymerase sliding clamp, <it>pcn</it>, and the flap endonuclease, <it>rad2</it>, 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.</p> <p>Conclusion</p> <p>The results showed that ten out of nineteen eukaryotic-type DNA replication genes are essential for <it>Halobacterium </it>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.</p>
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