Berquist Brian R, DasSarma Priya, DasSarma Shiladitya
University of Maryland Biotechnology Institute, Center of Marine Biotechnology, Baltimore, Maryland 21202, USA.
BMC Genet. 2007 Jun 8;8:31. doi: 10.1186/1471-2156-8-31.
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.
古菌中的信息传递系统，包括DNA复制机制的许多组成部分，与真核生物中的相似。古菌DNA复制基因的功能分配主要基于序列同源性和复制体组件的生化研究，但迄今为止进行的遗传研究很少。我们已经开发了一种易于处理的遗传系统，用于对模式嗜盐古菌盐生盐杆菌NRC-1中的基因进行敲除分析，并利用它来确定哪些DNA复制基因是必需的。
使用盐生盐杆菌NRC-1中的定向框内基因敲除方法，我们检测了19个预测参与DNA复制的基因。对与真核生物Orc1和Cdc6相关的大型嗜盐古菌Orc/Cdc6家族进行的初步生物信息学分析表明，在所有已测序的嗜盐古菌中，Orc/Cdc6蛋白有五个不同的进化枝保守存在。在盐生盐杆菌NRC-1的10个orc/cdc6基因中，仅发现两个是必需的，即大染色体上的orc10和小染色体pNRC200上的orc2。在三个复制型DNA聚合酶基因中，两个是必需的：染色体编码的B家族polB1和染色体编码的广古菌特异性D家族polD1/D2（在盐生盐杆菌NRC-1基因组序列中以前称为polA1/polA2）。pNRC200编码的B家族聚合酶polB2是非必需的。DNA复制起始和延伸因子的辅助基因，包括推定的复制解旋酶mcm、真核型DNA引发酶pri1/pri2、DNA聚合酶滑动夹pcn和瓣状核酸内切酶rad2，都是必需的。如果获得了敲除，则将靶向基因分类为非必需基因；如果基于统计分析和/或通过证明除了存在该基因的互补质粒拷贝外无法分离染色体敲除，则将其分类为必需基因。
结果表明，19个真核型DNA复制基因中有10个对盐生盐杆菌NRC-1是必需的，这与其对DNA复制的需求一致。必需基因编码10个Orc/Cdc6蛋白中的2个、3个DNA聚合酶中的2个、MCM解旋酶、2个DNA引发酶亚基、DNA聚合酶滑动夹和瓣状核酸内切酶。
