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. 2004 May 4;101(18):7046-51.
doi: 10.1073/pnas.0400656101. Epub 2004 Apr 23.

Three Replication Origins in Sulfolobus Species: Synchronous Initiation of Chromosome Replication and Asynchronous Termination

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Free PMC article

Three Replication Origins in Sulfolobus Species: Synchronous Initiation of Chromosome Replication and Asynchronous Termination

Magnus Lundgren et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Chromosome replication origins were mapped in vivo in the two hyperthermophilic archaea, Sulfolobus acidocaldarius and Sulfolobus solfataricus, by using microarray-based marker frequency analysis. Bidirectional replication was found to be initiated in near synchrony from three separate sites in both organisms. Two of the three replication origins in each species were located in the vicinity of a cdc6/orc1 replication initiation gene, whereas no known replication-associated gene could be identified near the third origin in either organism. In contrast to initiation, replication termination occurred asynchronously, such that certain replication forks continued to progress for >40 min after the others had terminated. In each species, all replication forks advanced at similar DNA polymerization rates; this was found to be an order of magnitude below that displayed by Escherichia coli and thus closer to eukaryotic elongation rates. In S. acidocaldarius, a region containing short regularly spaced repeats was found to hybridize aberrantly, as compared to the rest of the chromosome, raising the possibility of a centromere-like function.

Figures

Fig. 1.
Fig. 1.
Optical density measurements. (A) Batch culture of S. solfataricus grown into stationary phase. Sampling time points from exponential and stationary phase are indicated by arrows. (B) Batch culture of S. acidocaldarius. (C) Synchronization of an exponentially growing S. acidocaldarius batch culture. At an optical density of 0.1, the culture was split into two flasks, one of which was treated with acetic acid. After 4 h, the acetic acid was removed by centrifugation and fresh medium added. Open circles, culture treated with acetic acid; filled circles, untreated control.
Fig. 2.
Fig. 2.
MF distributions. (A) Ratio of hybridization signals from exponentially growing vs. stationary phase S. acidocaldarius cells. (B) Exponential growth vs. stationary phase for S. solfataricus.(C) Theoretical simulations (see Materials and Methods) of different replication initiation modes in S. acidocaldarius cell populations. The thick line denotes initiation from all three replication origins in each cell. The thin line shows initiation from a single origin, randomly chosen among the three.
Fig. 3.
Fig. 3.
Cell size and DNA content distributions from S. acidocaldarius synchronization experiment. Acetic acid was added to the culture at -265 min and removed at 0 min; samples for flow cytometry were removed at the indicated time points. Note the decreased average cell size after the 75-min time point, concomitant with the appearance of cells containing a single chromosome, demonstrating ongoing cell division.
Fig. 4.
Fig. 4.
(A) Flow cytometry DNA content (fluorescence) distributions from an S. acidocaldarius population progressing through the cell cycle in synchrony. (B) MF distributions from the same time points. (C) Progression of replication forks around the circular S. acidocaldarius chromosome.

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