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Comparative Study
. 2007 Apr 3;104(14):5806-11.
doi: 10.1073/pnas.0700206104. Epub 2007 Mar 28.

Extrachromosomal Element Capture and the Evolution of Multiple Replication Origins in Archaeal Chromosomes

Affiliations
Free PMC article
Comparative Study

Extrachromosomal Element Capture and the Evolution of Multiple Replication Origins in Archaeal Chromosomes

Nicholas P Robinson et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

In all three domains of life, DNA replication begins at specialized loci termed replication origins. In bacteria, replication initiates from a single, clearly defined site. In contrast, eukaryotic organisms exploit a multitude of replication origins, dividing their genomes into an array of short contiguous units. Recently, the multiple replication origin paradigm has also been demonstrated within the archaeal domain of life, with the discovery that the hyperthermophilic archaeon Sulfolobus has three replication origins. However, the evolutionary mechanism driving the progression from single to multiple origin usage remains unclear. Here, we demonstrate that Aeropyrum pernix, a distant relative of Sulfolobus, has two origins. Comparison with the Sulfolobus origins provides evidence for evolution of replicon complexity by capture of extrachromosomal genetic elements. We additionally identify a previously unrecognized candidate archaeal initiator protein that is distantly related to eukaryotic Cdt1. Our data thus provide evidence that horizontal gene transfer, in addition to its well-established role in contributing to the information content of chromosomes, may fundamentally alter the manner in which the host chromosome is replicated.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Characterization of the Aeropyrum pernix replication origins by two-dimensional (2D) gel electrophoresis. (A) Alignment of the UCM sequences located centrally in the two A. pernix and three Sulfolobus origins (St, Sulfolobus tokodaii; Sac, S. acidocaldarius; Ss, S. solfataricus). (B) Cartoon representing the A. pernix genome, indicating the position of the two origins relative to the initiator genes Cdc6-1 and Cdc6-2. (C and D) Representation of the two origin loci. Ovals denote the origin locations. Gene ID numbers are indicated; ORFs above the midline are transcribed left to right, and those below the line are transcribed from right to left. (E) Illustration of the species of replication intermediates detectable at the origins. (FK) 2D gel examination of the A. pernix origin loci. DNA isolated from asynchronous replicating A. pernix cells was digested with restriction enzymes to produce the fragments indicated in C and D and subjected to the 2D procedure. G and J display bubble arc intermediates that are indicative of an active bidirectional origin of replication within the central third of the restriction fragment.
Fig. 2.
Fig. 2.
A hybrid arrangement of Sulfolobus origin associated gene homologues is observed at the A. pernix oriC2 locus. Genes located at two S. solfataricus initiation sites, oriC1 and oriC3, are positioned together at the A. pernix oriC2. Homologous genes are denoted by color, and the gene ID numbers are indicated. Transfer-RNA genes (blue) and viral integration sites (black) are also highlighted.
Fig. 3.
Fig. 3.
Organization and orientation of the oriC3 loci in three Sulfolobus species. Colored hatching represents homologous regions of the genome in the three species. The direction of the hatching indicates the orientation of the homologous regions relative to the rest of the genome. A 21.5-kb intrafragment translocation is indicated by the yellow hatching. Origins are represented by ovals. The magnified segment of the S. acidocaldarius region illustrates the oriC3 proximal genes. S. solfataricus gene homologues are also displayed. ORF colors denote gene function: red, copG; green, WhiP; and yellow, probable stress response-related genes. HSP60*, thermosome α subunit belonging to the HSP60 family.
Fig. 4.
Fig. 4.
Organization of WhiP and its relationship to eukaryal Cdt1. (A) Sequence alignment of archaeal WhiP proteins [Sac, S. acidocaldarius (GenBank accession no. YP_256028); Sto, S. tokodaii (GenBank accession no. NP_377180); Mse, M. sedula (GenBank accession no. ZP_01600793); Sso, S. solfataricus (GenBank accession no. NP_342366); Ape, A. pernix (GenBank accession no. NP_148313); Hbu, H. butylicus (GenBank accession no. YP_001013395)] with human Cdt1 residues 296–546 (GenBank accession no. EAW66763) and yeast Saccharomyces cerevisiae Cdt1 residues 354–604 (GenBank accession no. NP_012580). The alignment was generated by using ClustalW and colored in Jalview (30). (B) Cartoon depicting the two conserved DNA-binding domains in the S. acidocaldarius WhiP ORF. These domains, of the “winged-helix” DNA-binding domain superfamily (SSF46785), were identified by using InterProScan (version 14.0; www.ebi.ac.uk/InterProScan).
Fig. 5.
Fig. 5.
The Winged-Helix Initiator Protein (WhiP) binds S. solfataricus oriC3. DNaseI footprinting analysis of WhiP, Cdc6-1, Cdc6-2, and Cdc6-3 interactions with oriC3 is shown. The position of previously described 12-bp inverted repeats (ir) and Sso0866 and Sso0867 ORFs are indicated at the side of the panel. (A) 0, 85, 100, and 130 nM WhiP on the oriC3 upper strand. (B and C) Cooperative effects of WhiP on Cdc6 binding on the oriC3 upper and lower strands, respectively. (B) Lanes 1, 3, 6, and 9: no protein; lanes 2, 4, 7, and 10: 130 nM WhiP, 500 nM Cdc6-1, 250 nM Cdc6-2, and 100 nM Cdc6-3, respectively; lanes 5, 8, and 11: 130 nM WhiP plus 500 nM Cdc6-1, 250 nM Cdc6-2, or 100 nM Cdc6-3, respectively. (C) Lanes 1, 4, 7, and 10: no protein; lanes 2, 3, 5, 8, and 11: 125 nM WhiP, 150 nM WhiP, 1,000 nM Cdc6-1, 200 nM Cdc6-2, and 75 nM Cdc6-3, respectively; lanes 6, 9, and 12: 150 nM WhiP plus 1,000 nM Cdc6-1, 200 nM Cdc6-2, or 75 nM Cdc6-3, respectively. (D) Cartoon summarizing the footprinting at oriC3. The open boxes labeled M denote regions of modification rather than discrete footprints.

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