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Comparative Study
. 2007 Feb 7;26(3):816-24.
doi: 10.1038/sj.emboj.7601529. Epub 2007 Jan 25.

Sister Chromatid Junctions in the Hyperthermophilic Archaeon Sulfolobus Solfataricus

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Free PMC article
Comparative Study

Sister Chromatid Junctions in the Hyperthermophilic Archaeon Sulfolobus Solfataricus

Nicholas P Robinson et al. EMBO J. .
Free PMC article

Abstract

Although the Archaea exhibit an intriguing combination of bacterial- and eukaryotic-like features, it is not known how these prokaryotic cells segregate their chromosomes before the process of cell division. In the course of our analysis of the third replication origin in the archaeon Sulfolobus solfataricus, we identify and characterise sister chromatid junctions in this prokaryote. This pairing appears to be mediated by hemicatenane-like structures, and we provide evidence that these junctions persist in both replicating and postreplicative cells. These data, in conjunction with fluorescent in situ hybridisation analyses, suggest that Sulfolobus chromosomes have a significant period of postreplicative sister chromatid synapsis, a situation that is more reminiscent of eukaryotic than bacterial chromosome segregation mechanisms.

Figures

Figure 1
Figure 1
Characterisation of the third origin of replication by 2D gel electrophoresis. (A) Schematic representation of the genomic organisation of the locus encompassing the third replication origin, oriC3, and two additional sites distal to the origin. Boxes depict ORFs; ORFs above the midline are transcribed from left to right and those below the line from right to left. The restriction endonuclease recognition sites producing the seven DNA species (C–I) utilised in this analysis are indicated. The triangles within the 192 bp noncoding region between ORFs Sso0866 and Sso0867 represent a 12 bp short inverted repeat motif located with the origin region. (B) Cartoon illustrating the three species of branched DNA molecule detectable at oriC3. (D–H) 2D gel dissection of the oriC3 locus. Genomic DNA, isolated from asynchronous replicating S. solfataricus cultures, was digested with restriction enzymes to produce the fragments indicated in panel A, and subjected to 2D gel electrophoresis. After transfer to nylon membrane, the DNA was hybridised with fragment-specific radiolabelled probes. Bubble-shaped replication intermediates are detectable in panels E and F, indicative of an active origin of replication. Spots located within the Y-shape intermediates in panels D, E and F demonstrate replication pausing at this locus. The prominent X-spikes evident at this locus represent joint molecules, and are strongest in the PvuII restriction fragment, centred on the origin (F). (C, I) 2D analysis of two additional sites distal to oriC3. X- shaped molecules remain detectable, but are considerably less abundant than at the origin. Prominent replication fork pause sites are detected in panels D, F and G and are indicated by arrowheads.
Figure 2
Figure 2
DNAseI footprint analysis of Cdc6-1, Cdc6-2 and Cdc6-3 binding to oriC3. MG denotes a Maxam Gilbert A+G sequencing ladder. The positions of the 12 bp inverted repeats and Sso0866 and Sso0867 ORFs are indicated at the side of the panels. (A) 0, 65, 130 and 260 nM Cdc6-2 on the oriC3 lower strand. (B) 0, 100, 125 and 160 nM Cdc6-3 on the oriC3 lower strand. (C) 0, 53.5, 105 and 210 nM Cdc6-2 on the oriC3 upper strand. (D) 0, 80, 93.5 and 112 nM Cdc6-3 on the oriC3 upper strand. (E) DNAseI footprinting of Cdc6-1 on the oriC3 upper strand. Lanes 1 and 2; 0 and 1100 nM Cdc6-1; lanes 3–6; 0, 550, 1100 and 2200 nM Cdc6-1 plus 112 nM Cdc6-3. The appearance of the Cdc6-1 footprint is coincidental with the loss of the Cdc6-3 protection at higher concentrations of Cdc6-1. (F) Cartoon summarising the footprinting at oriC3. It should be noted that when higher concentrations of Orc1/Cdc6s proteins are employed in footprinting assays, the binding of the Orc1/Cdc6s becomes nonspecific and nucleates cooperative assembly of higher order structures that extend away from the central specific zone of protection.
Figure 3
Figure 3
X-shaped molecules are present at all three replication origins and persist into the nonreplicating stationary phase. (A) Fluorescence histogram of an asynchronous replicating S. solfataricus culture (optical density (A600) of 0.3). The fluorescence values in the histogram feature two peaks corresponding to one and two copies (1C and 2C) of the genome, respectively. The histogram region between the two peaks includes those cells in DNA synthesis (S) phase. (B–D) 2D gel analysis of the three origins of replication using DNA isolated from the replicating population analysed in panel A (panel B depicts oriC1 (AccI digest), panel C illustrates oriC2 (BamHI/XcmI digest) and panel D demonstrates oriC3 (PvuII digest)). Bubble-, fork- and X-shaped molecules are present at each origin. (E) Fluorescence histogram of a stationary phase S. solfataricus culture. (F–H) 2D gel analysis of the three origin loci using DNA isolated from the stationary phase population (restriction fragments are identical to panels B–D). Although bubble-shaped molecules are no longer detectable, X- and Y-shaped species persist.
Figure 4
Figure 4
X- and Y-shaped structures are enhanced at both origin and nonorigin loci following UV treatment (200 J m−2 at 254 nm). (A–D) Neutral/neutral 2D agarose gel analysis of UV-treated and control DNA at OriC3 and a nonorigin region (655 454–658 591 bp). Standard neutral/neutral 2D agarose gel electrophoresis was performed as described previously (Robinson et al, 2004). (A) PvuII-digested OriC3 fragment—untreated (control) filter. (B) PvuII-digested OriC3 fragment after 200 J m−2 UV treatment. (C) BamHI/XcmI-digested nonorigin region—untreated (control) filter. (D) BamHI/XcmI-digested non-origin region after 200 J m−2 UV treatment. UV-treated and control DNA was prepared as described in Materials and methods.
Figure 5
Figure 5
Resolution of the origin-associated X-shaped species into linear molecules by branch migration assay. (A) Untreated (control) filter. DNA isolated from asynchronous replicating S. solfataricus culture was digested with PvuII and subjected to the standard 2D gel electrophoresis. The resulting membrane was hybridised with an oriC3-specific probe. (B, C) Branch migration of the X spikes at oriC3 in the absence or presence of magnesium ions, respectively. After digestion with PvuII, the genomic DNA was subjected to the first dimension electrophoresis. Gel slices excised from the first dimension were then incubated in branch migration buffer (either without (B) or with (C) magnesium ions) at 65°C for 2 h before separation in the second dimension. The X-spike is indicated by the letter ‘X' and its resolved product by ‘rX'. The prominent fork-stall site on the Y-arc is indicated by S and its resolution products by rS1 and rS2. Additionally, migration strengthens a signal due to X-shaped molecules arising from migration of species ‘S'; this spike is labelled rSX. (D) A diagram of the principal species detected in panels B and C. The diagrams indicate potential structures for species rS1, rS2, S and rSX.
Figure 6
Figure 6
FISH staining of S. solfataricus origins of replication in G1 and G2/M sorted populations, demonstrating chromosome pairing at G2 phase. (A) Fluorescence histogram of the G1 (1C) sorted population. (BD, F–H) Left-hand boxes exhibit confocal photomicrographs of FITC-labelled origins (oriC1, oriC2 and oriC3, respectively), counterstained with DAPI, in G1 (B–D) or G2/M (2C, panels F–H) sorted cells. Phase-contrast micrographs are displayed on the right-hand side of each panel. (E) FACS profile of the G2/M sorted population. (I) Enlarged images of single and double foci from oriC2-labelled cells. (J) Table summarising numbers of one, two and zero foci cells for each origin in both cell populations. (K) Histograms illustrating the FISH analysis data for each of the three origin-specific probes. In total, 1994 G1 sorted and 2587 G2/M sorted cells were counted, and scored into one of three categories, depending on whether an individual cell contained two, one or zero origin-specific spots. The histograms display the relative proportions of these three categories counted in the G1 or G2/M-sorted cells, for each of the three origins of replication. The individual cell counts are summarised in the table.

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