Subunit organization of Mcm2-7 and the unequal role of active sites in ATP hydrolysis and viability.
The Mcm2-7 (minichromosome maintenance) complex is a toroidal AAA(+) ATPase and the putative eukaryotic replicative helicase. Unlike a typical homohexameric helicase, Mcm2-7 contains six distinct, essential, and evolutionarily conserved subunits. Precedence to other AAA(+) proteins suggests that Mcm ATPase active sites are formed combinatorially, with Walker A and B motifs contributed by one subunit and a catalytically essential arginine (arginine finger) contributed by the adjacent subunit. To test this prediction, we used copurification experiments to identify five distinct and stable Mcm dimer combinations as potential active sites; these subunit associations predict the architecture of the Mcm2-7 complex. Through the use of mutant subunits, we establish that at least three sites are active for ATP hydrolysis and have a canonical AAA(+) configuration. In isolation, these five active-site dimers have a wide range of ATPase activities. Using Walker B and arginine finger mutations in defined Mcm subunits, we demonstrate that these sites similarly make differential contributions toward viability and ATP hydrolysis within the intact hexamer. Our conclusions predict a structural discontinuity between Mcm2 and Mcm5 and demonstrate that in contrast to other hexameric helicases, the six Mcm2-7 active sites are functionally distinct.
Bochman
Matthew L
ML
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Bell
Stephen P
SP
Schwacha
Anthony
A
D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone; D004265:DNA Helicases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D005656:Fungal Proteins; C489423:MCM5 protein, human; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004265:DNA Helicases; C069199:MCM2 protein, S cerevisiae