Reconstitution of the Mcm2-7p heterohexamer, subunit arrangement, and ATP site architecture.
The Mcm2-7p heterohexamer is the presumed replicative helicase in eukaryotic cells. Each of the six subunits is required for replication. We have purified the six Saccharomyces cerevisiae MCM proteins as recombinant proteins in Escherichia coli and have reconstituted the Mcm2-7p complex from individual subunits. Study of MCM ATPase activity demonstrates that no MCM protein hydrolyzes ATP efficiently. ATP hydrolysis requires a combination of two MCM proteins. The fifteen possible pairwise mixtures of MCM proteins yield only three pairs of MCM proteins that produce ATPase activity. Study of the Mcm3/7p ATPase shows that an essential arginine in Mcm3p is required for hydrolysis of the ATP bound to Mcm7p. Study of the pairwise interactions between MCM proteins connects the remaining MCM proteins to the Mcm3/7p pair. The data predict which subunits in the ATPase pairs bind the ATP that is hydrolyzed and indicate the arrangement of subunits in the Mcm2-7p heterohexamer.
Davey
Megan J
MJ
Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021, USA.
Indiani
Chiara
C
O'Donnell
Mike
M
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C085711:mcm5 protein, S pombe; C069199:MCM2 protein, S cerevisiae; C092802:MCM4 protein, S cerevisiae; C491088:MCM7 protein, S cerevisiae; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; C092300:mcm6 protein, S pombe