plication is an interesting subject in the research field of archaeal DNA replication. The main questions are how the initiation of replication from multiple origins is regulated and how the replication forks progress after the collision of two forks from opposite directions.
Figure 2. The oriC region in Pyrococcus genome.
3. How does Cdc6/Orc1 recognize oriC ?
important step in characterizing the initiation of DNA replication in Archaea is to understand how the Cdc6/Orc1 protein recognizes the oriC region. Based upon aminoacid sequence alignments, the archaeal Cdc6/Orc1 proteins belong to the AAA + family of proteins. The crystal structures of the Cdc6/Orc1 protein from Pyrobaculum aerophilum and one of the two Cdc6/Orc1 proteins, ORC2 from Aeropyrum pernix (the two homologs in this organism are called ORC1 and ORC2 by the authors) were determined. These Cdc6/Orc1 proteins consist of three structural domains. I and II adopt a fold found in the AAA + family proteins. A winged helix (WH) fold, which is present in a number of DNA binding proteins, is found in the domain III. There are four ORBs arranged in pairs on both sides of the DUE in the oriC region of A. pernix, and ORC1 binds to each ORB as a dimer. A mechanism was proposed in which ORC1 binds to all four ORBs to introduce a higher-order assembly for unwinding of the DUE with alterations in both topology and superhelicity. Furthermore, the crystal structures of S. solfataricus Cdc6-1 and Cdc6-3 (two of the three Cdc6/Orc1 proteins in this organism) forming a heterodimer bound to ori2 DNA (one of the three origins in this organism), and that of A. pernix ORC1 bound to an origin sequence were determined. These studies revealed that both the N-terminal AAA + ATPase domain (domain I + II) and C-terminal WH domain (domain III) contribute to origin DNA binding, and the structural information not only defined the polarity of initiator assembly on the origin but also indicated the induction of substantial distortion, which probably triggers the unwinding of the duplex DNA to start replication, into the DNA strands. These structural data also provided the detailed interaction mode between the initiator protein and the oriC DNA. analyses of the Methanothermobactor thermautotrophicus Cdc6-1 protein revealed the essential interaction between an arginine residue conserved in the archaeal Cdc6/Orc1 and an invariant guanine in the ORB sequence. P. furiosus Cdc6/Orc1 is difficult to purify in a soluble form. A specific site in the oriC to start unwinding in vitro , was identified using the protein prepared by a denaturation-renaturation procedure recently < i align="justify">.
As shown in Figure 2, the local unwinding site is about 670 bp away from the transition site between leading and lagging syntheses, which was determined earlier by an ...
