The sub-cellular localization of Sulfolobus DNA replication

Abstract

Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

Figure 1.

Sulfolobus acidocaldarius and S. solfataricus strains expressing FLAG-tagged TK from P. aerophilum incorporate BrdU into genomic DNA. (A) The TK-FLAG gene, expressed from a tf55α promoter, was integrated into the amyA locus of S. acidocaldarius MW001 using a ‘pop-in pop-out’ approach. Double crossover homologous recombination was used to integrate the TK-FLAG gene, expressed from an arabinose-inducible promoter, into the amyA locus of S. solfataricus PBL2025. The lacS marker confers growth to PBL2025 in lactose minimal media. The regions flanking the amyA gene are labelled amyA-up and amyA-down. (B) Western blot analysis of whole cell extracts from exponential cultures of parental strains (MW001 or PBL2025) and TK-FLAG integration strains (SacTK and SsoTK) using an anti-FLAG antibody, and anti-TBP antibody as a control. S. solfataricus cultures were grown in the absence or presence of 0.4% l-arabinose (l-Ara). (C) PCR amplification across the amyA locus using genomic DNA template generated 4.8 and 2.7 kb products in the parental strains MW001 and PBL2025, respectively, and 3.2 and 4.1 kb products in the mutant strains SacTK and SsoTK, respectively, indicating replacement of amyA with the tf55α-TK-FLAG constructs. (D) MW001 (dashed lines) and SacTK (solid lines) cells were grown in medium containing the indicated concentrations of BrdU and growth was monitored by A₆₀₀. Genomic DNA was extracted from 6 h samples, digested with PstI and separated by electrophoresis. DNA was transferred to a nitrocellulose membrane and probed with an anti-BrdU antibody. (E) PBL2025 (dashed lines) and SsoTK (solid lines) cells were grown in medium containing 0.4% l-Ara and the indicated concentrations of BrdU, and growth was monitored by A₆₀₀. Genomic DNA was extracted from 12 h samples, digested with EagI and separated by PFGE. DNA was transferred to a nitrocellulose membrane and probed with an anti-BrdU antibody.

Figure 2.

Sulfolobus acidocaldarius cells expressing FLAG-TK incorporate EdU into genomic DNA. (A) MW001 (dashed lines) and SacTK (solid lines) cells were grown in medium containing the indicated concentrations of EdU and growth was monitored by A₆₀₀. Genomic DNA was extracted from 24 h samples and digested with PstI. Incorporated EdU was fluorescently labelled using ‘click’ chemistry. Three hundred nanograms of each DNA sample were spotted onto a nitrocellulose membrane and visualized using a FLA5000 Phosphoimager. (B) Fluorescent microscopy of SacTK cells grown for 3 h with 100 µM EdU. After fixing with 2.5% paraformaldehyde, cellular DNA was fluorescently labelled using ‘click’ chemistry. Images show DIC, DAPI staining of DNA (blue), EdU-AlexaFluor488 (green) and merged images.

Figure 3.

Flow cytometry of S. acidocaldarius grown in the presence of nucleoside analogues. Fifty millilitre cultures containing the indicated amount of EdU (or BrdU where shown) were inoculated with S. acidocaldarius SacTK or MW001 to an OD₆₀₀ of 0.05 and grown for 3 h before performing flow cytometry.

Figure 4.

Localization of replicating DNA in synchronized S. acidocaldarius cells. SacTK cells were synchronized using the baby machine method and then incubated at 75°C for 20 min with 200 µM EdU to label newly synthesized DNA. Images show phase contrast, DAPI staining of DNA (blue), EdU-AlexaFluor488 (green) and merged images. (A) A representative image of cells and (B) representative images of cells scored as showing 1, 2 or 3 EdU foci are shown. (C) The number of EdU foci detected was counted (509 cells total) and the percentage of cells in each category is shown. Cell synchronization was assessed using flow cytometry, shown inset. (D) Using 2D slices, template circles encompassing 100 and 50% of the cell area were generated and EdU foci were assigned to the inner or outer 50% of the cell area (n = 145).

Figure 5.

Localization of replisomes in synchronized S. acidocaldarius cells. SacTK cells were synchronized and then grown at 75°C for 20, 40 or 60 min before fixing. Images show DIC, DAPI staining of DNA (blue), antibody labelling of PCNA1 (red; images were obtained in the FITC channel and false coloured red) and merged images. (A) A representative image of cells and (B) representative images of cells scored as showing 1–6 PCNA1 foci, or fully labelled, are shown. (C) Cell synchronization was assessed using flow cytometry. (D) The number of PCNA1 foci detected was counted from cells fixed at the indicated time after release, and the percentage of cells in each category is shown. (E) Synchronized SacTK cells were grown at 75°C for 15 min and then labelled with 200 µM EdU for 10 min before fixing. The number of EdU foci detected was counted (500 cells total) and the percentage of cells in each category is shown.