Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1
Open Access
- 8 June 2007
- journal article
- research article
- Published by Springer Science and Business Media LLC in BMC Genomic Data
- Vol. 8 (1), 31
- https://doi.org/10.1186/1471-2156-8-31
Abstract
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, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential. Using 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. The 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.Keywords
This publication has 39 references indexed in Scilit:
- SRP19 Is a Dispensable Component of the Signal Recognition Particle in ArchaeaJournal of Bacteriology, 2007
- Regulation of Mercury Resistance in the Crenarchaeote Sulfolobus solfataricusJournal of Bacteriology, 2006
- Characterization of a Methanosarcina acetivorans mutant unable to translate UAG as pyrrolysineMolecular Microbiology, 2005
- The Hyperthermophilic Euryarchaeota Pyrococcus abyssi Likely Requires the Two DNA Polymerases D and B for DNA ReplicationJournal of Molecular Biology, 2005
- Development of genetic methods and construction of a chromosomal glnK1 mutant in Methanosarcina mazei strain Gö1Molecular Genetics and Genomics, 2005
- Arsenic Resistance inHalobacteriumsp. Strain NRC-1 Examined by Using an Improved Gene Knockout SystemJournal of Bacteriology, 2004
- Regulation of Minichromosome Maintenance Helicase Activity by Cdc6Journal of Biological Chemistry, 2003
- An exosome‐like complex in Sulfolobus solfataricusEMBO Reports, 2003
- Identification of short ‘eukaryotic’ Okazaki fragments synthesized from a prokaryotic replication originEMBO Reports, 2003
- Common domains in the initiators of DNA replication inBacteria, ArchaeaandEukarya: combined structural, functional and phylogenetic perspectivesFEMS Microbiology Reviews, 2003