ABSTRACT
Minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. Studies have shown that the MCM complex from the thermoacidophilic euryarchaeon Thermoplasma acidophilum (TaMCM) has some properties not reported in other archaeal MCM helicases. Here, the biochemical properties of the TaMCM are studied. The protein binds single-stranded DNA, has DNA-dependent ATPase activity and ATP-dependent 3' --> 5' helicase activity. The optimal helicase conditions with regard to temperature, pH and salinity are similar to the intracellular conditions in T. acidophilum. It is also found that about 1,000 molecules of TaMCM are present per actively growing cell.
Subject(s)
Archaeal Proteins/metabolism , Chromosomes, Archaeal , Thermoplasma/metabolism , Adenosine Triphosphatases/metabolism , Base Sequence , DNA Primers , DNA Replication , DNA, Single-Stranded/metabolism , Dimerization , Enzyme Activation , Fluorescence Polarization , Hydrogen-Ion Concentration , Temperature , Thermoplasma/enzymology , Two-Hybrid System TechniquesABSTRACT
The MCM gene from the archaeon Halobacterium, with and without its intein, was cloned into an Escherichia coli expression vector, overexpressed and the protein was purified and antibodies were generated. The antibodies were used to demonstrate that in vivo only the processed enzyme, without the intein, could be detected.
ABSTRACT
The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. In most archaeal species studied, the interaction between MCM and the initiator protein, Cdc6, inhibits helicase activity. To date, the only exception is the helicase and Cdc6 proteins from the archaeon Thermoplasma acidophilum. It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity. It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM. Also, through the use of site-specific substitutions, and truncated and chimeric proteins, it was shown that an intact Cdc6 protein is required for this stimulation. ATP binding and hydrolysis by the Cdc6 protein is not needed for the stimulation. The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity.
