Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius

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 Ϸ88 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.Archaea ͉ cell cycle ͉ DNA replication

Biogeography of theSulfolobus islandicuspan-genome

“…The sets of genes contributing to this second pattern are primarily localized to a highly variable portion of each genome (Fig. 3, ring 4) that averages 695 kbp in length beginning Ϸ300 kbp from the origin in each strain [defined as 1 of 3 replication origins (57,58)], except in Y.N.15.51, which has had a large inversion (Fig. S1F).…”

Archaeal orthologs of Cdc45 and GINS form a stable complex that stimulates the helicase activity of MCM

“…This is the last region of S. acidocaldarius chromosome to be replicated (18). The ChIP signal from S phase cells indicates that replication forks have not yet progressed to this region in a significant number of cells (Fig.…”

“…qPCR was performed using 14 primer pairs for amplicons distributed across the SacCdc45 genome, including primers targeting the three origins of replication. MCM has previously been shown to be recruited to the S. acidocaldarius origins of replication at initiation of DNA replication (18), and as the presumed replicative helicase, MCM would be predicted to travel with the moving replication forks during S phase.…”

“…The procedure for ChIP assays is described in detail in SI Materials and Methods (26). ChIP was performed using anti-MCM or anti-cmyc antibodies, as described previously (18). The resulting purified DNA was quantified by qPCR.…”

“…SacCdc45 strains were synchronized using the baby machine method (18). After formaldehyde-fixation, ChIP was performed using anti-MCM and anti-myc antibodies from cells fixed in G1/early S-phase, mid-S-phase and G2 phase (Fig.…”