Coker James A, DasSarma Priya, Capes Melinda, Wallace Tammitia, McGarrity Karen, Gessler Rachael, Liu Jingfang, Xiang Hua, Tatusov Roman, Berquist Brian R, DasSarma Shiladitya
University of Maryland Biotechnology Institute, Center of Marine Biotechnology, 701 E. Pratt Street, Suite 236, Baltimore, MD 21202, USA.
J Bacteriol. 2009 Aug;191(16):5253-61. doi: 10.1128/JB.00210-09. Epub 2009 Jun 5.
The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and -200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.
模式嗜盐古菌嗜盐栖热袍菌NRC-1的类真核生物DNA复制系统由一条环状染色体以及两个大型大质粒或微型染色体pNRC100和pNRC200编码。我们之前通过遗传分析表明，编码Orc-Cdc6复制起始蛋白的10个基因中的2个（orc2和orc10）是必需的，而位于高度保守的自主复制序列oriC1附近的第三个基因（orc7）对细胞活力并非必需。在此，我们使用全基因组标记频率分析（MFA），发现了多个峰值，表明存在多个复制起点。最大的染色体峰值位于orc7（oriC1）和orc10（oriC2）附近，而染色体外元件上的最大峰值在pNRC100和pNRC200中均靠近orc9（oriP1），在pNRC200中靠近orc4（oriP2）。对含有不同组合染色体orc基因的缺失菌株进行MFA分析表明，oriC1处的复制起始需要orc7，但不需要orc6和orc8。通过复制起始点分析确定了oriC1处的起始位点，发现其在一个富含AT的元件内和附近呈发散状定位，该元件两侧可能是Orc结合位点。oriC1区域、Orc结合位点和orc7基因直系同源物在所有已测序的嗜盐古菌中均保守。对orc基因进行系列缺失导致构建了一个最小菌株，该菌株不仅含有orc2和orc10，还含有orc9。我们的结果表明，该模式系统中的复制比之前认为的更具吸引力且更复杂。我们从嗜盐古菌基因组的复制策略和进化角度讨论了这些结果。
