Published online 9 September 2004
Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved
Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus
Department of Molecular and Cell Biology, 227 Hildebrand Hall #3206, University of California Berkeley, Berkeley, CA 94720, USA
* To whom correspondence should be addressed. Tel: +1 510 643 9483; Fax: +1 510 643 9290; Email: jmberger{at}uclink4.berkeley.edu
Received July 21, 2004; Revised and Accepted August 23, 2004
Archaeal cell division cycle protein 6 (Cdc6)/Origin Replication Complex subunit 1 (Orc1) proteins share sequence homology with eukaryotic DNA replication initiation factors but are also structurally similar to the bacterial initiator DnaA. To better understand whether Cdc6/Orc1 functions in an eukaryotic or bacterial-like manner, we have characterized the interaction of two Cdc6/Orc1 paralogs (mthCdc6-1 and mthCdc6-2) with the replication origin from Methanothermobacter thermoautotrophicus. We show that while both proteins display a low affinity for a small dsDNA of random sequence, mthCdc6-1 binds tightly to a short duplex containing a single copy of a 13 bp sequence that is repeated throughout the origin. Surprisingly, sequence comparisons show that this 13 bp sequence is a minimized version of the Origin Recognition Box element found in many euryarchaeotal origins. Analysis of mthCdc6-1 mutants demonstrates that the helix–turn–helix motif in the winged-helix domain mediates the interaction with this sequence. Association of both mthCdc6/Orc1 paralogs with the duplex containing the minimized Origin Recognition Box fits to an independent binding sites model, but their interaction with longer DNA ligands is cooperative. Together, our data provide the first detailed biophysical characterization of the association of an archaeal DNA replication initiator with its origin. Our observations also indicate that the origin-binding properties of Cdc6/Orc1 proteins closely resemble those of bacterial DnaA.
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