Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus.

ABSTRACT

The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing. Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea. The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue. The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro. The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA. Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA. The purified Orc/Cdc6 homologue was also able to bind DNA. Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates. Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap. The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer.