Biochemical Analysis of a DNA Replication Origin in the Archaeon Aeropyrum pernix

We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple...

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Bibliographic Details
Published in:Journal of molecular biology 2006-10, Vol.363 (2), p.355-369
Main Authors: Grainge, Ian, Gaudier, Martin, Schuwirth, Barbara S., Westcott, Sarah L., Sandall, Jane, Atanassova, Neli, Wigley, Dale B.
Format: Article
Language:English
Subjects:
Aeropyrum - genetics
Aeropyrum - metabolism
Aeropyrum pernix
archaea
Archaeal Proteins - genetics
Archaeal Proteins - metabolism
Base Sequence
DNA - chemistry
DNA - metabolism
DNA Footprinting
DNA replication
Hydroxyl Radical - metabolism
Models, Molecular
Molecular Sequence Data
Nucleic Acid Conformation
Origin Recognition Complex - genetics
Origin Recognition Complex - metabolism
Protein Binding
Protein Subunits - genetics
Protein Subunits - metabolism
Replication Origin
Single-Strand Specific DNA and RNA Endonucleases - metabolism
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Summary:We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple origin recognition box (ORB) sites that we show to be a replication origin. At this origin, there are four ORB elements disposed either side of an A + T-rich region. An ORC1 protein dimer binds at each of these ORB sites. Once all four ORB sites have bound ORC1 protein, there is a transition to a higher-order assembly with a defined alteration in topology and superhelicity. Furthermore, after this transition, the A + T-rich region becomes sensitive to digestion by DNase I and P 1 nuclease, revealing that the transition promotes distortion of the DNA in this region, presumably as a prelude to loading of MCM helicase.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2006.07.076