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. 2007 May 18;3(5):e77.
doi: 10.1371/journal.pgen.0030077. Epub 2007 Apr 5.

Genetic and Physical Mapping of DNA Replication Origins in Haloferax Volcanii

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Free PMC article

Genetic and Physical Mapping of DNA Replication Origins in Haloferax Volcanii

Cédric Norais et al. PLoS Genet. .
Free PMC article

Abstract

The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. DNA Replication Origin on Contig 454 Is on Chromosomes pHV1 and pHV4
(A) Sequence features of the ARS element isolated from genomic libraries of WR340 DNA. Coordinates of plasmid inserts generated by HpaII and AciI digestion are shown (including insert in pTA194), in addition to the minimal ARS element determined by AciI digestion (in pTA250) or PCR amplification (in pCN12). Numbering refers to TIGR contig 454 (pHV1). (B) Sequence of repeats found at the intergenic region of the pHV1/4 replication origin (correspond to numbered arrows in Figure 1A). Orientation is indicated by arrows (righthand side) and conserved sequences are shaded. Six of the 13 repeats feature a complete ORB element (boxed), while a core mini-ORB element is conserved in all repeats. The sequence motif found in repeats surrounding the DUE is indicated by the dashed box. (C) Southern blot of PFG of intact DNA from strains H53 and H230, probed with HpaII ARS insert from pTA194, or intergenic region replaced by trpA in pTA266 (see Figure 1D). (D) Intergenic region of ori-pHV1/4 was replaced by trpA marker by using the deletion construct pTA266. H. volcanii H53 was transformed with pTA266 to generate H220, which was used to derive the pHV1/4 origin deletion strain H230. Predicted fragment sizes of StuI digest are indicated. (E) StuI digest of genomic DNA from strains H53, H220, and H230, probed with DNA flanking the intergenic region. The band indicated * represents episomal DNA carrying the pHV1/4 origin, resulting from excision of the integrated plasmid.
Figure 2
Figure 2. DNA Replication Origin on the Main Chromosome: oriC1
(A) Sequence features of the ARS element isolated from genomic libraries of H230 DNA, including selected genes (see text for details). Coordinates of plasmid inserts generated by AciI digestion are shown (including insert in pTA313), in addition to the minimal ARS element in pTA441 and pCN11. See Figure 1A for key. Numbering refers to TIGR contig number 455. Main chromosome, Chr. (B) Above the line are sequences of repeats found at the intergenic region of the H. volcanii chromosomal replication origin (correspond to numbered arrows in Figure 2A). Below the line are sequences of repeats found at other (presumed) archaeal origins. The species and relevant cdc6/orc1 genes are H. marismortui cdc6–4 (Hmar-1–2), Halobacterium sp. NRC-1 orc7 (NRC1-1-2), Natronomonas pharaonis cdc6–1 (Npha-1–2), S. solfataricus cdc6–1 (Sso-1–2), and P. abyssi cdc6 (Pab-1–4). The orientation is indicated by arrows and conserved positions are shaded. Among halophilic archaea, repeats surrounding the primary DUE feature a longer consensus sequence (Halo-ORB, boxed), which contains the core mini-ORB and “G-string” elements also found in other archaea, plus a halophile-specific “G-string.” (C) Southern blot of PFG of DNA from strain H53, probed with the AciI ARS insert from pTA313.
Figure 3
Figure 3. ori-pHV1/4 ARS Plasmids Are Dominant to, but Less Stable Than, oriC-1 ARS Plasmids
(A) H112 was transformed with a mixture of 0.5 μg each of pTA194 and pTA313. Crude DNA was isolated from 23 transformants and a Southern blot probed with pyrE2 sequences. (B) Transformants 1–12 were propagated by restreaking on selective Hv-Ca agar. DNA was isolated and probed as above. (C) Transformants 1–12 were restreaked on nonselective Hv-YPC agar. DNA was isolated and probed as above. (D) H112 containing pTA250 or pTA441 was grown in nonselective Hv-YPC broth. At regular intervals aliquots were plated on selective (Hv-Ca) and nonselective (Hv-YPC) agar, to determine the fraction of uracil+ cells.
Figure 4
Figure 4. Nucleotide Disparity Curves
(A) Nucleotide disparity curves of H. volcanii DS2 genome sequence. Positions of the 14 cdc6/orc1 genes and five replication origins identified in this work are indicated. A putative prophage sequence with high A + T content results in a sharp peak of nucleotide disparity for the main chromosome (indicated by dotted line), but does not coincide with any ARS element isolated here. Nucleotide skews are calculated using the ZPLOTTER program (http://tubic.tju.edu.cn/zcurve). (B) GC disparity curves of the chromosome of four halophilic archaea, a halophilic bacterium, and a non-halophilic archaeon. Chromosome coordinates have been offset to begin at H. sp. NRC-1 orc7, N. pharaonis cdc6, H. marismortui cdc6–4, H. walsbyi cdc6–1, S. ruber dnaA, and P. abyssi cdc6 genes, which are assumed or proven to be associated with the replication origins. The halophilic bacterium and non-halophilic archaeon were used as a control.
Figure 5
Figure 5. A Second DNA Replication Origin on the Main Chromosome: oriC2
(A) Sequence features of the second origin from main chromosome. Coordinates of the 9.3-kb NotI genomic fragment cloned in pTA416 and the 2.4-kb intergenic region cloned in pTA612 are shown. See Figure 1A for key. Numbering refers to TIGR contig number 455. Main chromosome, Chr. (B) Sequence of repeats found at the intergenic region of oriC2 (numbered arrows in Figure 5A). The orientation is indicated by arrows and conserved sequences are shaded. (C) Southern blot of PFG of DNA from strain H53, probed with intergenic region cloned in pTA612.
Figure 6
Figure 6. Additional ARS Elements Predicted by Nucleotide Composition
(A) Sequence features of replication origin on pHV3. Coordinates of 693-bp intergenic region cloned in pCN26 are shown. See Figure 1A for key. Numbering refers to TIGR contig number 453 (pHV3). (B) Sequence of repeats found at the intergenic region of the pHV3 origin (numbered arrows in Figure 6A). The orientation is indicated by arrows and conserved sequences are shaded. (C) Southern blot of PFG of DNA from strain H53, probed with intergenic region cloned in pCN26. (D) Sequence features of ARS element in contig number 452. Coordinates of 592-bp intergenic region cloned in pCN27 are shown. Numbering refers to TIGR contig number 452 (pHV4). (E) Sequence of repeats found at intergenic region of contig number 452 ARS element (numbered arrows in Figure 6D). (F) Southern blot of PFG of DNA from strain H53, probed with intergenic region cloned in pCN27.
Figure 7
Figure 7. Mapping of Initiation Sites at the Replication Origins
(A) Primer extension reactions were performed with enriched replicating intermediate DNA fragments and separated on a denaturing polyacrylamide gel. Corresponding primers are indicated on top of the gels (see Table S1 for details), arrows refer to the 5′ end of amplification products and transition points are indicated by bold arrows. (B) Primers and detected initiation sites along the sequence. The positions of DUEs, the associated cdc6/orc1 gene on the right, and the upstream open reading frame on the left are shown for each replication origin. Dots indicate weak amplification products obtained with control templates (linearized plasmid DNA carrying the replication origins, isolated from E. coli cells).

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