The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum ΔH contains DNA helicase activity

Abstract

In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric (Ϸ10 3 kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3-to-5 ATPdependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.replication ͉ ORC1 protein ͉ Cdc6 protein T he minichromosome maintenance (MCM) genes encode a family of proteins first identified by their essential role in the maintenance of ARS-containing minichromosomes in Saccharomyces cerevisiae (1). MCM homologues have been identified in all eukaryotes from yeast to mammals and have highly conserved amino acid sequences (2, 3). The MCM family consists of at least six distinct polypeptides (MCM2-7). In addition to forming a heterohexameric structure, the MCM proteins are capable of forming various subcomplexes in vitro (4-8). Although the MCM proteins were shown to play an important role in the initiation and elongation phases of DNA replication, the precise functions of the MCM complexes are not yet fully understood (reviewed in refs. 3, 9, and 10). In vitro studies have revealed several biochemical properties of the MCM proteins. A heterotrimeric complex of human (h) MCMs (MCM4, -6, and -7) that forms a dimeric structure contains ATP-dependent DNA helicase activity, binds to single-stranded DNA, and possesses DNA-dependent ATPase activity (11). It was also demonstrated that the hMCM2 protein interacts with the hMCM4, -6, and -7 complex to form the heterotetrameric complex, hMCM2, -4, -6, and -7, which is devoid of the activities associated with the hMCM4, -6, and -7 complex (11, 12). Identical observations have been made with MCM proteins isolated from Schizosaccharomyces pombe (spMCM; J.-K.L. and J.H., unpublished observations).In addition to genetic studies that demonstrated the essential role of the MCM proteins in DNA replication, biochemical studies showed that these proteins are assembled onto origins of replication in S. cerevisiae (13,14). Furthermore, the proteins appear to interact genetically and physically with the origin recognition complex (ORC) and other proteins that participate in the initiation of DNA replication such as . These genetic data obtained from yeast, together with the in vitro biochemical properties, have led to the suggestion that the MCM proteins may function as the replicative helicase in eukaryotes, similar to role of the bacterial DnaB protein in Escherichia coli replication and the large tumor antigen (T Ag) in the replication of simian virus 40 (SV40) .Archaea, the third domain of life (21), are believed to replicate DNA in a eukaryotic-like f...

Crystal structure of a near-full-length archaeal MCM: Functional insights for an AAA+ hexameric helicase

“…However, some archaeal MCM proteins, such as Sulfolobus solfataricus MCM (ssoMCM) and Methanothermobacter thermautotrophicus MCM (mtMCM), are encoded by a single mcm gene. Both ssoMCM and mtMCM can form homooligomers (9)(10)(11). The N-terminal region is poorly conserved among MCM proteins from archaea to eukaryotes.…”

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…(54)(55)(56) Electron microscopy has shown the MCM dodecamer to have a ring-like structure formed by two opposed hexamers. (55) Consistent with this observation, the crystal structure of the N-terminal domain of MtMCM protein reveals a dodecamer formed by a double-hexamer, with the shape of a dumbbell and a 118 Å long channel running from side to side.…”

“…Different degrees of helicase activity have also been reported in MCM proteins from S. pombe, (52) Xenopus (53) and an archaeobacteria in which a single MCM homolog forms a double hexamer. (54)(55)(56) It should be noted that helicase activity and processivity might be underrated in experiments with purified MCM proteins, because a replicative helicase is likely to function in the context of a bigger complex containing one or several regulatory activities. (57,58) Possible mechanisms for MCM helicase loading and mode of action are discussed later.…”

Biochemical Characterization of the Methanothermobacter thermautotrophicus Minichromosome Maintenance (MCM) Helicase N-terminal Domains

“…Filter Binding Assay-Nitrocellulose filter binding assays were carried out by incubating 0.1, 0.3, 0.9, and 2.7 pmol of proteins (as monomers) at 60°C for 10 min in 15 l of buffer containing 20 mM Hepes-NaOH (pH 7.5), 10 mM MgCl 2 , 2 mM dithiothreitol, 100 g/ml bovine serum albumin, 1 mM ATP, and 50 fmol of 5Ј 32 P-labeled oligonucleotide N120T; 5Ј-(GTTT) 10 CGCTGCTCTGCCTCCCGCTGCTCT-GCCTCCACTCAGCTCCCTGGCACAGCCTGTCCTGGCACAGGCTG-TCCACGTCTGGC-3Ј (2500 cpm/fmol). After incubation, the mixture was filtered through an alkaline-washed nitrocellulose filter (HA 0.45 mm; Millipore) (22), which was then washed with 20 mM Hepes-NaOH (pH 7.5).…”