Crystal structure of the hexameric replicative helicase RepA of plasmid RSF101011Edited by R. Huber

Niedenzu, Timo; Röleke, D.; Bains, Gabrielle; Scherzinger, Eberhard

doi:10.1006/jmbi.2000.4398

Cited by 90 publications

(142 citation statements)

References 39 publications

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“…Significantly Rep40 is structurally related to the AAA ϩ family of motor proteins (ATPases associated with diverse cellular activities) (16). Characteristic of the AAA ϩ architecture as well as that of other oligomeric NTPases is the presence of an "arginine finger" that translates ATP hydrolysis into intermolecular conformational changes in the context of a functional oligomeric complex (21,23). Mutation of the conserved arginine finger in Rep abrogates helicase and ATPase function, consistent with a hexameric model for Rep40 (see Fig.…”

supporting

confidence: 54%

Residues within the B′ Motif Are Critical for DNA Binding by the Superfamily 3 Helicase Rep40 of Adeno-associated Virus Type 2

Yoon-Robarts¹,

Blouin²,

Bleker

et al. 2004

Journal of Biological Chemistry

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We have recently published the crystal structure of the adeno-associated virus type 2 superfamily 3 (SF3) helicase Rep40. Although based on its biochemical properties it is unlikely that Rep40 plays a central role as a replicative helicase the involvement of this motor protein in DNA packaging has recently been demonstrated. Here we focused our attention on residues that fall within and adjacent to the B motif of SF3 helicases that directly interact with single-stranded DNA during translocation of the motor protein. In vitro, alanine substitution at positions Lys-404 or Lys-406 abrogated the ability of the protein to interact with single-stranded DNA as demonstrated by electrophoretic mobility shift assay and fluorescence anisotropy, and accordingly these mutants could not unwind a partially duplex DNA substrate. Despite this loss of helicase activity, basal ATPase activity in these mutants remained intact. However, unlike the wild-type protein, K404A and K406A ATPase activity was not stimulated by DNA. As predicted, disruption of motor activity through interference with DNA binding resulted in an inability of Rep40 to package adeno-associated virus DNA in a tissue culture-based assay. Taken together, we characterized, for the first time in an SF3 helicase family member, residues that are directly involved in single-stranded DNA binding and that are critical for the Rep motor activity. Based on our findings we propose B as the signature motif of SF3 helicases that is responsible for the complex interactions required for the coupling of DNA binding and ATP hydrolysis.

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supporting

confidence: 54%

Residues within the B′ Motif Are Critical for DNA Binding by the Superfamily 3 Helicase Rep40 of Adeno-associated Virus Type 2

Yoon-Robarts¹,

Blouin²,

Bleker

et al. 2004

Journal of Biological Chemistry

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“…ORF86 encodes a protein with homology to the replicative helicase RepA of gram-negative plasmids belonging to the DnaB-like helicase family. These proteins unwind double-stranded DNA into single-stranded intermediates fueled by nucleoside triphosphate hydrolysis (29). The putative protein encoded by ORF88 shows high homology to the T1p22 helicase, another protein involved in DNA unwinding and synthesis (37).…”

Section: Resultsmentioning

confidence: 99%

Broad-Host-Range Yersinia Phage PY100: Genome Sequence, Proteome Analysis of Virions, and DNA Packaging Strategy

et al. 2008

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PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage Aa⌽23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.

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“…This prediction has been vindicated by recent determinations of the crystal structures of two hexameric helicases that are distant relatives of DnaB: the RSF1010 RepA protein (132,174) and the Cterminal helicase domain of T7 gene 4 protein (152,157). The overall structures of these hexamers are similar.…”

Section: Structures Of Dnab and Related Hexameric Helicasesmentioning

confidence: 99%

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

Neylon

Kralicek

Hill

et al. 2005

Microbiol Mol Biol Rev

134

144

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The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process

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Crystal structure of the hexameric replicative helicase RepA of plasmid RSF101011Edited by R. Huber

Cited by 90 publications

References 39 publications

Residues within the B′ Motif Are Critical for DNA Binding by the Superfamily 3 Helicase Rep40 of Adeno-associated Virus Type 2

Residues within the B′ Motif Are Critical for DNA Binding by the Superfamily 3 Helicase Rep40 of Adeno-associated Virus Type 2

Broad-Host-Range Yersinia Phage PY100: Genome Sequence, Proteome Analysis of Virions, and DNA Packaging Strategy

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

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