Showing papers by "Stephen D. Bell published in 2011"

Molecular determinants of origin discrimination by Orc1 initiators in archaea

Abstract: Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA+) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA+ domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA+ domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.

Replication termination and chromosome dimer resolution in the archaeon Sulfolobus solfataricus.

Abstract: Archaea of the genus Sulfolobus have a single-circular chromosome with three replication origins. All three origins fire in every cell in every cell cycle. Thus, three pairs of replication forks converge and terminate in each replication cycle. Here, we report 2D gel analyses of the replication fork fusion zones located between origins. These indicate that replication termination involves stochastic fork collision. In bacteria, replication termination is linked to chromosome dimer resolution, a process that requires the XerC and D recombinases, FtsK and the chromosomal dif site. Sulfolobus encodes a single-Xer homologue and its deletion gave rise to cells with aberrant DNA contents and increased volumes. Identification of the chromosomal dif site that binds Xer in vivo, and biochemical characterization of Xer/dif recombination revealed that, in contrast to bacteria, dif is located outside the fork fusion zones. Therefore, it appears that replication termination and dimer resolution are temporally and spatially distinct processes in Sulfolobus.

The role of the DNA sliding clamp in Okazaki fragment maturation in archaea and eukaryotes.

Abstract: Efficient processing of Okazaki fragments generated during discontinuous lagging-strand DNA replication is critical for the maintenance of genome integrity. In eukaryotes, a number of enzymes co-ordinate to ensure the removal of initiating primers from the 5′-end of each fragment and the generation of a covalently linked daughter strand. Studies in eukaryotic systems have revealed that the co-ordination of DNA polymerase δ and FEN-1 (Flap Endonuclease 1) is sufficient to remove the majority of primers. Other pathways such as that involving Dna2 also operate under certain conditions, although, notably, Dna2 is not universally conserved between eukaryotes and archaea, unlike the other core factors. In addition to the catalytic components, the DNA sliding clamp, PCNA (proliferating-cell nuclear antigen), plays a pivotal role in binding and co-ordinating these enzymes at sites of lagging-strand replication. Structural studies in eukaryotic and archaeal systems have revealed that PCNA-binding proteins can adopt different conformations when binding PCNA. This conformational malleability may be key to the co-ordination of these enzymes9 activities.

DNA replication: archaeal oriGINS.

Abstract: GINS is an essential eukaryotic DNA replication factor that is found in a simplified form in Archaea. A new study in this issue of BMC Biology reveals the first structure of the archaeal GINS complex. The structure reveals the anticipated similarity to the previously determined eukaryotic complex but also has some intriguing differences in the relative disposition of subunit domains.

Archaeal RNA polymerase: the influence of the protruding stalk in crystal packing and preliminary biophysical analysis of the Rpo13 subunit

Abstract: We review recent results on the complete structure of the archaeal RNAP (RNA polymerase) enzyme of Sulfolobus shibatae . We compare the three crystal forms in which this RNAP packs (space groups P 2 1 2 1 2 1 , P 2 1 2 1 2 and P 2 1 ) and provide a preliminary biophysical characterization of the newly identified 13-subunit Rpo13. The availability of different crystal forms for this RNAP allows the analysis of the packing degeneracy and the intermolecular interactions that determine this degeneracy. We observe the pivotal role played by the protruding stalk composed of subunits Rpo4 and Rpo7 in the lattice contacts. Aided by MALLS (multi-angle laser light scattering), we have initiated the biophysical characterization of the recombinantly expressed and purified subunit Rpo13, a necessary step towards the understanding of Rpo139s role in archaeal transcription.

The interplay of DNA binding, ATP hydrolysis and helicase activities of the archaeal MCM helicase.

Abstract: The MCM (minichromosome maintenance) proteins of archaea are widely believed to be the replicative DNA helicase of these organisms. Most archaea possess a single MCM orthologue that forms homo-multimeric assemblies with a single hexamer believed to be the active form. In the present study we characterize the roles of highly conserved residues in the ATPase domain of the MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results identify a potential conduit for communicating DNA-binding information to the ATPase active site.