Replication Initiation Point Mapping: Approach and Implications

ABSTRACT 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.

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Where it all starts: eukaryotic origins of DNA replication

Journal of Cell Science ◽

10.1242/jcs.114.4.643 ◽

2001 ◽

Vol 114 (4) ◽

pp. 643-651 ◽

Cited By ~ 1

Author(s):

A.K. Bielinsky ◽

S.A. Gerbi

Keyword(s):

Dna Replication ◽

Binding Sites ◽

Transcription Initiation ◽

Origin Recognition Complex ◽

Replication Initiation ◽

Cell Cycle Regulators ◽

Initiation Point ◽

The Past ◽

Eukaryotic Genomes ◽

Made In

Chromosomal origins of DNA replication in eukaryotic cells not only are crucial for understanding the basic process of DNA duplication but also provide a tool to analyze how cell cycle regulators are linked to the replication machinery. During the past decade much progress has been made in identifying replication origins in eukaryotic genomes. More recently, replication initiation point (RIP) mapping has allowed us to detect start sites for DNA synthesis at the nucleotide level and thus to monitor replication initiation events at the origin very precisely. Beyond giving us the precise positions of start sites, the application of RIP mapping in yeast and human cells has revealed a single, defined start point at which replication initiates, a scenario very reminiscent of transcription initiation. More importantly, studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site. Therefore, in our pursuit of identifying ORC-binding sites in higher eukaryotes, RIP mapping may lead the way.

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