Double Hexamer Structure Of The Archaeal Helicase MCM From Methanobacterium Thermoautotrophicum

Abstract

Hexameric DNA helicases are key enzymes in the replicative machinery. They are also an example of flexible proteins which undergo conformational changes related to their function. The minichromosome maintenance factor MCM of Methanobacterium thermoautotrophicum (mtMCM) is at present the best characterized archaeal replicative helicase, as well as a useful experimental model for the more complex eukaryotic helicases. Biochemical and crystallographic evidence indicate that the double hexamer is the functional form of mtMCM, but previous EM reports have detected assembly as single heptamer, single hexamer, or other stoichiometries.In our studies, we have observed not only 6-fold and 7-fold structures, but also open rings and double rings in the same wild type mtMCM preparation. This is an indication of polymorphism in the assembly of mtMCM, and possibly of equilibrium between these various forms. It is not clear at present, what is the functional relevance of each of the structural arrangements, although a hexameric form would correlate best with the presence of six MCM components in the eukaryotic MCM2-7 complex. The presence of open ring forms, reported here for the first time, suggests that loading of mtMCM onto DNA might be achieved through a ring opening mechanism. Such an MCM loading mechanism would be similar to that proposed for the T7 helicase or the bacterial Rho terminator, and different to that of the SV40 large T antigen, where monomers assemble around the DNA to form the hexameric rings.We also present the first three-dimensional reconstruction of the MCM double hexamer from negatively stained samples. The map allows direct observation of the dodecameric complex for the first time, and highlights characteristics similar to those found for SV40 large T antigen, such as the existence of side channels in each hexamer.