Biochemical characterization of the minichromosome maintenance (MCM) protein of the crenarchaeote Aeropyrum pernix and its interactions with the origin recognition complex (ORC) proteins.
Replication in archaea is carried out by proteins that are homologues of eukaryotic counterparts. However, the archaeal systems tend to be much simpler with fewer different genes encoding the core functions than in eukaryotic counterparts. In many archaea, there is a single minichromosome maintenance (MCM) homologue, presumed to be the replicative helicase and between one and three origin recognition complex (ORC) homologues involved in binding to the replication origins. Here we describe the cloning and characterization of the MCM protein from the crenarchaeote Aeropyrum pernix. Like other eukaryotic and archaeal MCM proteins, it is found to be an ATP-dependent DNA helicase, and the putative active site residues involved in ATP binding and hydrolysis are confirmed by mutation. Deletion of the N-terminal 256 amino acids yielded a protein with higher ATPase activity in the absence of DNA and retained robust helicase activity. Interactions with the ORC proteins of A. pernix were examined, and it was found that both ORC homologues could inhibit the helicase activity of MCM. Further it was found that ORC2 could autophosphorylate in the presence of ATP and more remarkably could phosphorylate MCM in a species-specific manner.
Atanassova
Neli
N
Cancer Research UK Clare Hall Laboratories, The London Research Institute, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3LD, UK.
Grainge
Ian
I
D041522:Aeropyrum / Q000737:chemistry* / Q000235:genetics; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D051738:Origin Recognition Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D040681:Structural Homology, Protein
D019843:Archaeal Proteins; D051738:Origin Recognition Complex