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 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 P1 nuclease, revealing that the transition promotes distortion of the DNA in this region, presumably as a prelude to loading of MCM helicase.
Grainge
Ian
I
Cancer Research UK, Clare Hall Laboratories, The London Research Institute, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3LD, UK.
Gaudier
Martin
M
Schuwirth
Barbara S
BS
Westcott
Sarah L
SL
Sandall
Jane
J
Atanassova
Neli
N
Wigley
Dale B
DB
D041522:Aeropyrum / Q000235:genetics / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D017665:Hydroxyl Radical / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D018741:Replication Origin; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D019843:Archaeal Proteins; D051738:Origin Recognition Complex; D021122:Protein Subunits; D017665:Hydroxyl Radical; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases