ORCID Profile
0000-0002-8474-6592
Current Organisation
Indiana University Bloomington
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Publisher: Proceedings of the National Academy of Sciences
Date: 28-10-2008
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 ≈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.
Publisher: Oxford University Press (OUP)
Date: 08-02-2012
DOI: 10.1093/NAR/GKS217
Publisher: Wiley
Date: 24-11-2008
DOI: 10.1111/J.1365-2958.2008.06500.X
Abstract: Bacteria that have a circular chromosome with a bidirectional DNA replication origin are thought to utilize a 'replication fork trap' to control termination of replication. The fork trap is an arrangement of replication pause sites that ensures that the two replication forks fuse within the terminus region of the chromosome, approximately opposite the origin on the circular map. However, the biological significance of the replication fork trap has been mysterious, as its inactivation has no obvious consequence. Here we review the research that led to the replication fork trap theory, and we aim to integrate several recent findings that contribute towards an understanding of the physiological roles of the replication fork trap. Likely roles include the prevention of over-replication, and the optimization of post-replicative mechanisms of chromosome segregation, such as that involving FtsK in Escherichia coli.
Publisher: Wiley
Date: 26-11-2010
Publisher: Oxford University Press (OUP)
Date: 13-02-2004
DOI: 10.1093/NAR/GKH259
Publisher: Elsevier BV
Date: 06-2006
Publisher: American Society for Microbiology
Date: 12-2007
DOI: 10.1128/JB.01016-07
Abstract: In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.JMB.2009.02.027
Abstract: The Escherichia coli chromosome contains two opposed sets of unidirectional DNA replication pause (Ter) sites that, according to the replication fork trap theory, control the termination of chromosome replication by restricting replication fork fusion to the terminus region. In contrast, a recent hypothesis suggested that termination occurs at the dif locus instead. Using two-dimensional agarose gel electrophoresis, we examined DNA replication intermediates at the Ter sites and at dif in wild-type cells. Two definitive signatures of site-specific termination--specific replication fork arrest and converging replication forks--were clearly detected at Ter sites, but not at dif. We also detected a significant pause during the latter stages of replication fork convergence at Ter sites. Quantification of fork pausing at the Ter sites in both their native chromosomal context and the plasmid context further supported the fork trap model.
Publisher: Springer New York
Date: 2019
DOI: 10.1007/978-1-4939-9492-2_1
Abstract: Members of the archaeal domain of life that lack homologs of actin and tubulin ide by binary fission in a process that is dependent upon orthologs of eukaryotic ESCRT components. Many of these archaeal organisms are hyperthermophilic acidophiles with unique cell wall structures, which create technical challenges for performing traditional cell biological techniques. Here, we describe the "baby machine" method for synchronizing microorganisms at high temperatures in order to study cell cycle-related processes. We also provide details for fixing, permeabilizing, and staining archaeal cells and ESCRT assemblies for observation by light microscopy.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Stephen Bell.