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. 2003 Feb;4(2):154-8.
doi: 10.1038/sj.embor.embor732.

Identification of Short 'Eukaryotic' Okazaki Fragments Synthesized From a Prokaryotic Replication Origin

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Free PMC article

Identification of Short 'Eukaryotic' Okazaki Fragments Synthesized From a Prokaryotic Replication Origin

Fujihiko Matsunaga et al. EMBO Rep. .
Free PMC article

Abstract

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.

Figures

Figure 1
Figure 1
Unmasking assay showing short replication intermediates in Pyrococcus abyssi and Sulfolobus acidocaldarius. The 5′-OH groups of RNA-primed replication intermediates were selectively exposed by alkaline treatment. 'Unmasked' 5′-OH ends of replication intermediates were labelled with T4 polynucleotide kinase and [γ-32P]ATP, and run on an alkaline agarose gel. A control experiment without alkaline treatment is also shown for each experiment. The positions of size markers (100, 200 and 500 nucleotides (nt)) are shown on the left.
Figure 2
Figure 2
Direct labelling of RNA on the 5′ termini of replication intermediates with vaccinia virus guanylyltransferase, an enzyme responsible for messenger RNA capping. (A) The 5′ ends of RNA primers attached to P. abyssi replication intermediates were capped with radioactive GTP and samples were fractionated on a sequencing gel (lane 2). Lane 3 contains the same reaction as lane 2, except that total DNA was treated with the restriction endonuclease EcoRI before analysis, confirming that nascent strands were efficiently denatured from template strands. Lane 1, size marker (10-base ladder; Gibco-BRL) labelled with [γ-32P]ATP using T4 polynucleotide kinase. (B) Schematic diagram of the archaeal Okazaki fragment. The smallest replication intermediate detected (10 nt) is in accordance with the minimum length of RNA primers detected in vitro (Liu et al., 2001).
Figure 3
Figure 3
RIP mapping (for a detailed principle of the assay see the recent review by Bielinsky & Gerbi (2001)) revealed the frequent initiation of Okazaki fragments in the P. abyssi oriC. (A) RIP mapping of the 'top' and 'bottom' strands with the F1 or R2 primer (see Fig. 4A), respectively. Arrows indicate the transition point (TP). (B) Discontinuous replication on the top strand was analysed with the F2 primer. Sequencing reactions were run side by side on a denaturing polyacrylamide gel to map initiation points. RI, replication intermediates.
Figure 4
Figure 4
Detailed structure of the P. abyssi OriC. (A) Summary of initiation sites for DNA synthesis detected by RIP mapping. Stronger bands on the gels are shown by longer black arrows, and weaker bands by shorter black arrows. The long inverted-repeat and short 13-bp repeats are shown by open arrows and open arrowheads, respectively, together with the cdc6/orc1 gene and a DNA-unwinding element (DUE). Primers used for RIP mapping of the top strand (F1 and F2) and the bottom strand (R1 and R2) are also indicated (red arrows). (B) Nucleotide sequence of a region containing the transition points (shown by arrows). The long inverted-repeat is shown in red capitals. (C) Conserved long inverted-repeats in Pyrococcus species. The consensus sequence is indicated at the bottom. Pab, P. abyssi; Pfu, P. furiosus; Pho, P. horikosii.

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