Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius

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Abstract
The "baby machine" provides a means of generating synchronized cultures of minimally perturbed cells. We describe the use of this technique to establish the key cell-cycle parameters of hyperthermophilic archaea of the genus Sulfolobus. The 3 DNA replication origins of Sulfolobus acidocaldarius were mapped by 2D gel analysis to near 0 (oriC2), 579 (oriC1), and 1,197 kb (oriC3) on the 2,226-kb circular genome, and we present a direct demonstration of their activity within the first few minutes of a synchronous cell cycle. We also detected X-shaped DNA molecules at the origins in log-phase cells, but these were not directly associated with replication initiation or ongoing chromosome replication in synchronized cells. Whole-genome marker frequency analyses of both synchronous and log-phase cultures showed that origin utilization was close to 100% for all 3 origins per round of replication. However, oriC2 was activated slightly later on average compared with oriC1 and oriC3. The DNA replication forks moved bidirectionally away from each origin at approximate to 88 bp per second in synchronous culture. Analysis of the 3 Orc1/Cdc6 initiator proteins showed a uniformity of cellular abundance and origin binding throughout the cell cycle. In contrast, although levels of the MCM helicase were constant across the cell cycle, its origin localization was regulated, because it was strongly enriched at all 3 origins in early S phase.