PubMed Health⌕ Search

Biomedical subjects

Stephen D Bell

Publications and source records attributed to Stephen D Bell.

At least 19 recordsLinked to original sources
PubMed2024

Capturing chromosome conformation in Crenarchaea.

While there is a considerable body of knowledge regarding the molecular and structural biology and biochemistry of archaeal information processing machineries, far less is known about the nature of the substrate for these machineries-the archaeal nucleoid. In this article, we will describe recent advances in our understanding of the three-dimensional organization of the chromosomes of model organisms in the crenarchaeal phylum.

Chromosomes, Archaeal↗
PubMed2006

A novel archaeal regulatory protein, Sta1, activates transcription from viral promoters.

While studying gene expression of the rudivirus SIRV1 in cells of its host, the hyperthermophilic crenarchaeon Sulfolobus, a novel archaeal transcriptional regulator was isolated. The 14 kDa protein, termed Sulfolobus transcription activator 1, Sta1, is encoded on the host chromosome. Its activating effect on transcription initiation from viral promoters was demonstrated in in vitro transcription experiments using a reconstituted host system containing the RNA polymerase, TATA-binding protein (TBP) and transcription factor B (TFB). Most pronounced activation was observed at low concentrations of either of the two transcription factors, TBP or TFB. Sta1 was able to bind viral promoters independently of any component of the host pre-initiation complex. Two binding sites were revealed by footprinting, one located in the core promoter region and the second approximately 30 bp upstream of it. Comparative modeling, NMR and circular dichroism of Sta1 indicated that the protein contained a winged helix-turn-helix motif, most probably involved in DNA binding. This strategy of the archaeal virus to co-opt a host cell regulator to promote transcription of its genes resembles eukaryal virus-host relationships.

Amino Acid Sequence↗
PubMed2006

Structure of an archaeal PCNA1-PCNA2-FEN1 complex: elucidating PCNA subunit and client enzyme specificity.

The archaeal/eukaryotic proliferating cell nuclear antigen (PCNA) toroidal clamp interacts with a host of DNA modifying enzymes, providing a stable anchorage and enhancing their respective processivities. Given the broad range of enzymes with which PCNA has been shown to interact, relatively little is known about the mode of assembly of functionally meaningful combinations of enzymes on the PCNA clamp. We have determined the X-ray crystal structure of the Sulfolobus solfataricus PCNA1-PCNA2 heterodimer, bound to a single copy of the flap endonuclease FEN1 at 2.9 A resolution. We demonstrate the specificity of interaction of the PCNA subunits to form the PCNA1-PCNA2-PCNA3 heterotrimer, as well as providing a rationale for the specific interaction of the C-terminal PIP-box motif of FEN1 for the PCNA1 subunit. The structure explains the specificity of the individual archaeal PCNA subunits for selected repair enzyme 'clients', and provides insights into the co-ordinated assembly of sequential enzymatic steps in PCNA-scaffolded DNA repair cascades.

Archaeal Proteins↗
PubMed2006

GINS, a central nexus in the archaeal DNA replication fork.

In eukaryotes, the GINS complex is essential for DNA replication and has been implicated as having a role at the replication fork. This complex consists of four paralogous GINS subunits, Psf1, Psf2, Psf3 and Sld5. Here, we identify an archaeal GINS homologue as a direct interaction partner of the MCM helicase. The core archaeal GINS complex contains two subunits that are poorly conserved homologues of the eukaryotic GINS subunits, in complex with a protein containing a domain homologous to the DNA-binding domain of bacterial RecJ. Interaction studies show that archaeal GINS interacts directly with the heterodimeric core primase. Our data suggest that GINS is important in coordinating the architecture of the replication fork and provide a mechanism to couple progression of the MCM helicase on the leading strand with priming events on the lagging strand.

Amino Acid Sequence↗
PubMed2006

The chromosome replication machinery of the archaeon Sulfolobus solfataricus.

In the three domains of life, the archaea, bacteria, and eukarya, there are two general lineages of DNA replication proteins: the bacterial and the eukaryal/archaeal lineages. The hyperthermophilic archaeon Sulfolobus solfataricus provides an attractive model for biochemical study of DNA replication. Its relative simplicity in both genomic and biochemical contexts, together with high protein thermostability, has already provided insight into the function of the more complex yet homologous molecules of the eukaryotic domain. Here, we provide an overview of recent insights into the functioning of the chromosome replication machinery of S. solfataricus, focusing on some of the relatively well characterized core components that act at the DNA replication fork.

Archaeal Proteins↗
PubMed2006

Influence of chromatin and single strand binding proteins on the activity of an archaeal MCM.

The mini-chromosome maintenance (MCM) complex is the presumptive replicative helicase in archaea and eukaryotes. In archaea, the MCM is a homo-multimer, in eukaryotes a heterohexamer composed of six related subunits, MCM 2-7. Biochemical studies using naked DNA templates have revealed that archaeal MCMs and a sub-complex of eukaryotic MCM 4, 6 and 7 have 3' to 5' helicase activity. Here, we investigate the influence of the major chromatin proteins, Alba and Sul7d, of Sulfolobus solfataricus (Sso) on the ability of the MCM complex to melt partial duplex DNA substrates. In addition, we test the effect of Sso SSB on MCM activity. We reveal that Alba represents a formidable barrier to MCM activity and further demonstrate that acetylation of Alba alleviates repression of MCM activity.

Acetylation↗
PubMed2006

DNA replication in the archaea.

The archaeal DNA replication machinery bears striking similarity to that of eukaryotes and is clearly distinct from the bacterial apparatus. In recent years, considerable advances have been made in understanding the biochemistry of the archaeal replication proteins. Furthermore, a number of structures have now been obtained for individual components and higher-order assemblies of archaeal replication factors, yielding important insights into the mechanisms of DNA replication in both archaea and eukaryotes.

Archaea↗
PubMed2005

Structure of the heterodimeric core primase.

Primases are DNA-dependent RNA polymerases that synthesize the oligoribonucleotide primers essential to DNA replication. In archaeal and eukaryotic organisms, the core primase is a heterodimeric enzyme composed of a small and a large subunit. Here we report a crystallographic and biochemical analysis of the core primase from the archaeon Sulfolobus solfataricus. The structure provides the first three-dimensional description of the large subunit and its interaction with the small subunit. The evolutionary conservation of amino acids at the protein-protein interface implies that the observed mode of subunit association is conserved among archaeal and eukaryotic primases. The orientation of the large subunit in the core primase probably excludes its direct involvement in catalysis. Modeling of a DNA-RNA helix together with structure-based site-directed mutagenesis provides insight into the mechanism of template DNA binding and RNA primer synthesis.

Amino Acid Sequence↗
PubMed2005

Organization of the archaeal MCM complex on DNA and implications for the helicase mechanism.

The homomultimeric archaeal mini-chromosome maintenance (MCM) complex serves as a simple model for the analogous heterohexameric eukaryotic complex. Here we investigate the organization and orientation of the MCM complex of the hyperthermophilic archaeon Sulfolobus solfataricus (Sso) on model DNA substrates. Sso MCM binds as a hexamer and slides on the end of a 3'-extended single-stranded DNA tail of a Y-shaped substrate; binding is oriented so that the motor domain of the protein faces duplex DNA. Two candidate beta-hairpin motifs within the MCM monomer have partially redundant roles in DNA binding. Notably, however, conserved basic residues within these motifs have nonequivalent roles in the helicase activity of MCM. On the basis of these findings, we propose a model for the mechanism of the helicase activity of MCM and note parallels with SV40 T antigen.

Adenosine Triphosphatases↗
PubMed2005

Characterization of an archaeal family 4 uracil DNA glycosylase and its interaction with PCNA and chromatin proteins.

We describe the characterization of a family 4 UDG1 (uracil DNA glycosylase) from the crenarchaeote Sulfolobus solfataricus. UDG1 is found to have a marked preference for substrates containing a G:U base pair over either A:U or single-stranded uracil-containing DNA substrates. UDG1 is found to interact with the sliding clamp PCNA (proliferating cell nuclear antigen), and does so by a conserved motif in the C-terminus of the protein. S. solfataricus has a heterotrimeric PCNA, and only one of the subunits, PCNA3, interacts with UDG1. We have been unable to detect any stimulation of UDG activity by PCNA, in contrast with the observed effects of PCNA on a number of DNA metabolic enzymes. However, analysis of the effects of Sulfolobus chromatin proteins on UDG1 leads us to propose a mechanistic basis for coupling UDG1 to the replication fork.

Bacterial Proteins↗
PubMed2005

Sir2 and the acetyltransferase, Pat, regulate the archaeal chromatin protein, Alba.

The DNA binding affinity of Alba, a chromatin protein of the archaeon Sulfolobus solfataricus P2, is regulated by acetylation of lysine 16. Here we identify an acetyltransferase that specifically acetylates Alba on this residue. The effect of acetylation is to lower the affinity of Alba for DNA. Remarkably, the acetyltransferase is conserved not only in archaea but also in bacteria where it appears to play a role in metabolic regulation. Therefore, our data suggest that S. solfataricus has co-opted this bacterial regulatory system to generate a rudimentary form of chromatin regulation.

Acetyltransferases↗
PubMed2005

The promiscuous primase.

DNA primases are essential for the initiation of DNA replication and progression of the replication fork. Recent phylogenetic analyses coupled with biochemical and structural studies have revealed that the arrangement of catalytic residues within the archaeal and eukaryotic primase has significant similarity to those of the Pol X family of DNA-repair polymerases. Furthermore, two additional groups of enzymes, the ligase/primase of the bacterial nonhomologous end-joining machinery and a putative replicase from an archaeal plasmid have shown striking functional and structural similarities to the core primase. The promiscuous nature of the archaeal primases suggests that these proteins might have additional roles in DNA repair in the archaea.

Catalytic Domain↗
PubMed2005

Archaeal transcriptional regulation--variation on a bacterial theme?

There is now an increasing body of data available on the DNA-binding properties of several putative transcriptional regulators in the Archaeal domain of life. The evidence points to simple models of promoter occlusion or facilitated recruitment of basal machinery for repressors and activators, respectively. However, little is known about the co-factor requirements, in vivo mechanisms and targets of many of these regulators. It is anticipated that the application of post-genomic technologies will begin to shed light on this fascinating area.

Archaea↗
PubMed2005

Origins of DNA replication in the three domains of life.

Replication of DNA is essential for the propagation of life. It is somewhat surprising then that, despite the vital nature of this process, cellular organisms show a great deal of variety in the mechanisms that they employ to ensure appropriate genome duplication. This diversity is manifested along classical evolutionary lines, with distinct combinations of replicon architecture and replication proteins being found in the three domains of life: the Bacteria, the Eukarya and the Archaea. Furthermore, although there are mechanistic parallels, even within a given domain of life, the way origins of replication are defined shows remarkable variation.

Archaea↗
PubMed2004

The heterodimeric primase of the hyperthermophilic archaeon Sulfolobus solfataricus possesses DNA and RNA primase, polymerase and 3'-terminal nucleotidyl transferase activities.

A eukaryotic-type primase was identified in the crenarchaeon Sulfolobus solfataricus. The two-subunit DNA-dependent primase, termed PriSL, was purified following co-expression of the subunits in Escherichia coli and its activity was characterised. PriSL was capable of utilising both ribonucleotides and deoxyribonucleotides for primer synthesis in the presence of natural, or synthetic, single-stranded DNA. A broad distribution of products was detected, ranging from dinucleotides to DNA molecules in excess of 7 kb and RNA up to 1 kb in length. However, PriSL had a significantly higher affinity for ribonucleotides than for deoxyribonucleotides. Using site-directed mutagenesis, two aspartate residues crucial for nucleic acid synthesis and residues important for the binding of free nucleotides were identified. In addition to the primase and polymerase activities, we reveal that the primase possesses a template-independent 3'-terminal nucleotidyl transferase activity.

Amino Acid Sequence↗