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. 2014 Feb;42(4):2282-94.
doi: 10.1093/nar/gkt1214. Epub 2013 Nov 22.

Multiple Replication Origins With Diverse Control Mechanisms in Haloarcula Hispanica

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Free PMC article

Multiple Replication Origins With Diverse Control Mechanisms in Haloarcula Hispanica

Zhenfang Wu et al. Nucleic Acids Res. .
Free PMC article

Abstract

The use of multiple replication origins in archaea is not well understood. In particular, little is known about their specific control mechanisms. Here, we investigated the active replication origins in the three replicons of a halophilic archaeon, Haloarcula hispanica, by extensive gene deletion, DNA mutation and genome-wide marker frequency analyses. We revealed that individual origins are specifically dependent on their co-located cdc6 genes, and a single active origin/cdc6 pairing is essential and sufficient for each replicon. Notably, we demonstrated that the activities of oriC1 and oriC2, the two origins on the main chromosome, are differently controlled. A G-rich inverted repeat located in the internal region between the two inverted origin recognition boxes (ORBs) plays as an enhancer for oriC1, whereas the replication initiation at oriC2 is negatively regulated by an ORB-rich region located downstream of oriC2-cdc6E, likely via Cdc6E-titrating. The oriC2 placed on a plasmid is incompatible with the wild-type (but not the ΔoriC2) host strain, further indicating that strict control of the oriC2 activity is important for the cell. This is the first report revealing diverse control mechanisms of origins in haloarchaea, which has provided novel insights into the use and coordination of multiple replication origins in the domain of Archaea.

Figures

Figure 1.
Figure 1.
Single deletions of candidate replication origins and their adjacent cdc6 genes. (A) Schematic diagram of the candidate ori-cdc6 origin region (not to scale), including the origin region and its adjacent cdc6 gene. (B) Summary of the knockout results: (+), knockouts were obtained for these targets; (−), deletions of these targets were not obtained; (+#), knockout of cdc6A gene was extremely difficult, which resulted in tiny colonies with a significant growth defect. The origins on different replicons (main chromosome, minichromosome and megaplasmid pHH400) are separated by dotted lines.
Figure 2.
Figure 2.
Genome-wide mapping of the chromosomal replication origins in H. hispanica wild-type and deletion strains. (A) MFA for the chromosome of the H. hispanica wild-type strain. The ratios of marker DNA hybridization signals of exponential to stationary phase are plotted against chromosome position (kilobase). The approximate locations of oriC1 and oriC2, which are proximal to cdc6A and cdc6E (indicated with arrows), respectively, are indicated with vertical lines. (B) Active replication origins on the chromosome of H. hispanica. The start position of the chromosome is reset from the location of the haloarchaeal conserved oriC1 origin [indicated by a round dot in (A)]. The approximate positions of the cdc6 genes are indicated, and the active cdc6-associated replication origins (oriC1 and oriC2) on the chromosome are in bold. The GC skew of the chromosome is represented by the inner circle. (C) The use of replication origins in chromosomal origin- and cdc6-deletion strains (ΔoriC1, ΔoriC2 and Δcdc6E) was monitored via MFA.
Figure 3.
Figure 3.
Specific recognition of replication origins by distinct Cdc6 homologs. (A) Schematic diagram of the ori-cdc6 origin (not to scale) and the location of the ori and ori-cdc6 deletions. Plasmids harboring an ori region only (pOC-A) or an ori-cdc6 region (pOC-B) for each replication origin were constructed and subjected to ARS activity assays. Corresponding Δori and Δori-cdc6 strains were used as transformation hosts. (B) Summary of the ARS activity assays. Plus (+) and minus (−) signs represent ARS activity and no ARS activity, respectively.
Figure 4.
Figure 4.
ARS plasmid containing oriC2 is incompatible with the H. hispanica wild-type strain. (A) Transformations with the pOC1-A and pOC2-A plasmids into the H. hispanica wild-type strain. Transformation with pOC2-A into ΔoriC2 strain was used as a control. Transformant number per microgram of DNA was quantified from three independent experiments. (B) Southern blot analysis of plasmid integration and autonomous replication led to Mevr colonies, which are indicated with grey and black arrows, respectively. Lane P represents the purified plasmid, which was used as an input control. The results were quantified for 20 transformants from two independent experiments.
Figure 5.
Figure 5.
Dissection of cis elements for replication initiation at oriC1. (A) DNA sequence of the minimal size of oriC1 (oriC1m). The ORB elements are boxed, and the extended halophile-specific ‘G-string’ elements are shaded (I and II). The G-rich inverted repeat located inside the two ORB elements is indicated with orientation arrows (III and IV). Primers for truncation analysis are indicated with bent arrows (oriC1mF and oriC1mR for oriC1m; D1F and oriC1mR for D1; oriC1mF and D2R for D2). (B) Sequence alignment of ORB elements. C-ORB represents a classic ORB element identified in archaeal origins. The ‘G-string’ and extended halophile-specific ‘G-string’ elements are indicated. (C) Transformations with plasmids constructed with a truncated or mutant oriC1 origin into the ΔoriC1 strain: oriC1m represents the minimal size of the oriC1 origin; D1 and D2 denote the deletion of the left and right ORB element, respectively; SMI to SMIV denote mutants for the four G-rich sites as in (A), respectively (CCCCCC to TTTTTT in I, GGGGGGGG to AAAAAAAA in II, GGGGAGGGGG to AAAAGAAAAA in III and CCCCCTCCCC to TTTTTCTTTT in IV). Colonies were observed after 7–8 days at 37°C, and the transformation efficiency and mean colony size were quantified from three independent experiments.
Figure 6.
Figure 6.
Negative control of replication origin activity at oriC2. (A) Knockout analyses and ARS assays of the oriC2 region. The ORB elements found in the intergenic regions flanking the cdc6E gene are indicated with numbered arrowheads. The yes (y) and no (n) signs in the knockout analyses indicate that knockouts were obtained (y) or not obtained (n). The plus (+) and minus (−) signs indicate ARS activity and no ARS activity, respectively. (B, D) Transformations with pOC2-B into ΔoriC2L and ΔoriC2-cdc6E (B), and pOC2-B and pOC2l into ΔoriC2L (D). Transformants were observed after 7–8 days at 37°C, and the transformation efficiency and mean colony size were quantified from three independent experiments. (C, E) Southern blot analysis of ARS plasmids in transformants as in (B) and (D) using a bla gene probe. The cells were quantified via OD measurements, and genomic DNA was used as a loading control. For each transformation, three colonies were repeated for Southern blot analysis, and the mean concentration of episomal plasmids was quantified from three independent experiments.
Figure 7.
Figure 7.
Growth curves of chromosomal origin deletion strains determined under different conditions. H. hispanica cultures were grown under standard conditions (37°C, 20% NaCl) (A): (left) growth at 37°C with shaking at 200 rpm; (right) Equal amounts of serial dilutions of exponentially growing cells (OD600 = ∼1.5) were spotted on AS-168 (20% NaCl) supplemented with uracil and grown for 7 days at 37°C. For salinity, the lowest salt concentration (16% NaCl) in which H. hispanica can survive (B) and a saturated salt concentration (C) were chosen. For temperature conditions, cultures were grown in nutrient-rich AS-168 medium at low (D) and high (E) temperatures.

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