Polymorphism and Double Hexamer Structure in the Archaeal Minichromosome Maintenance (MCM) Helicase from Methanobacterium thermoautotrophicum

Yacob Gómez-Llorente,Ryan J Fletcher,Xiaojiang S Chen,José M Carazo,Carmen San Martín

doi:10.1074/jbc.m509760200

Abstract

Methanobacterium thermoautotrophicum minichromosome maintenance complex (mtMCM), a cellular replicative helicase, is a useful model for the more complex eukaryotic MCMs. Biochemical and crystallographic evidence indicates that mtMCM assembles as a double hexamer (dHex), but previous electron microscopy studies reported only the presence of single heptamers or single hexamers (Pape, T., Meka, H., Chen, S., Vicentini, G., Van Heel, M., and Onesti, S. (2003) EMBO Rep. 4, 1079-1083; Yu, X., VanLoock, M. S., Poplawski, A., Kelman, Z., Xiang, T., Tye, B. K., and Egelman, E. H. (2002) EMBO Rep. 3, 792-797). Here we present the first three-dimensional electron microscopy reconstruction of the full-length mtMCM dHex in which two hexamers contact each other via the structurally well defined N-terminal domains. The dHex has obvious side openings that resemble the side channels of LTag (large T antigen). 6-fold and 7-fold rings were observed in the same mtMCM preparation, but we determined that assembly as a double ring favors 6-fold structures. Additionally, open rings were also detected, which suggests a direct mtMCM loading mechanism onto DNA.

Highlights

Throughout nature, replication of DNA begins with the ordered assembly of a number of proteins at the replication origin
Structural Polymorphism—The Methanobacterium thermoautotrophicum minichromosome maintenance complex (mtMCM) protein complex expressed and purified from E. coli behaved as a dodecamer in gel filtration chromatography (Fig. 1A), which is consistent with prior reports [5,6,7]
Previous EM studies have shown that the archaeal helicase mtMCM is able to adopt three different oligomeric arrangements, namely hexameric rings, heptameric rings, and helical polymers (10 –12)

Summary

Introduction

Throughout nature, replication of DNA begins with the ordered assembly of a number of proteins at the replication origin. LTag assembles as a head-to-head oriented dHex on the viral origin of replication (ori), with the C-terminal helicase domains forming the outer tiers of the cylinder and the DNA binding domains in the equator [14, 15].

Results

Conclusion