Human Mcm10 DNA replication factor

Elena Orlova
Andrei Okorokov
Julie Hodgkinson

Structure of initiation factor Mcm10

The DNA replication initiation factor Mcm10 is a conserved, abundant nuclear protein critical for origin firing. During the transition from pre-replicative complexes to pre-initiation complexes Mcm10 recruitment to replication origins is required for providing a physical link between the Mcm2-7 DNA helicase and DNA polymerases. The structure taken together with biochemical data suggests that this important protein exploits its architecture as a docking module for assembly of the molecular machinery required for initiation of eukaryotic DNA replication.

Human Mcm10 is a 98 kDa protein of 874 amino acids which contains a CCCH-type zinc-finger motif within its central part that appears to be conserved among homologues from yeast to mammals. Mcm10 interacts with a large number of replication initiation factors, yet it shows low homology across species and lacks significant similarities to other proteins at sequence level.

In this study we used electron microscopy and single particle analysis to determine the structure of full-length human Mcm10 protein. The Mcm10 molecule is a ring-shaped hexamer with large central and smaller lateral channels and a system of inner chambers. The 3D map of the protein was obtained at 16 resolution at 0.5 threshold of the Fourier shell correlation function. The overall shape of the Mcm10 molecule is a double-layered ring assembly with a 6-fold symmetry. The height of the molecule is 120 and the diameter is of 160

Secondary structure prediction revealed two large regions of the Mcm10 sequence which could be identified as independent domains. The first region is formed by ~250 aa residues (220-470) with a predicted secondary structure represented primarily by -strands. This region contains a conserved CCCH-type zinc-finger motif. There is a close similarity between the predicted secondary structure for this region of Mcm10 and the atomic structure of the N-terminal domain of the M. thermoautotrophicum MCM DNA helicase (mtMCM), including the positioning of the Zn-fingers in both proteins. The second region of the Mcm10 protein (640-874 aa) is predicted to be mainly a-helical. The region has eight conserved cystein residues that are clustered in two groups, thus potentially forming at least one additional zinc-finger in the 782-835 aa residues region. The secondary structure prediction for the C-terminal part of Mcm10 demonstrates close similarity to the architecture of the SV40 large T-antigen (SV40 LTag) C-terminal domain. We therefore used the atomic structures of mtMCM and SV40 LTag (PDB entries 1N25 and 1LTL) for docking into our human Mcm10 3D map.

Comparison of the secondary structure prediction for the Mcm10 protein sequence with (A) crystal structure and secondary structure prediction of mtMCM and (B) SV40 LTag. Red rectangular boxes represent a-helices, while blue arrowheads represent -strands. The secondary structure assigned to the crystal structures of mtMCM and SV40 LTag correlates well with the predicted structure of Mcm10 in the 240-480 aa and 640-870 aa regions respectively (highlighted by pink and blue boxes). Zn-binding clusters are indicated by yellow boxes. (C) The central slice of the structure with fitted SV40 LTag and mtMCM domains. Zn atoms are depicted as yellow spheres and indicated by arrows. (D) The top tier of the Mcm10 3D map at threshold 1s with fitted atomic coordinates of SV40 LTag (in blue). (E) mtMCM N-terminal domain (in red) fitted into the bottom tier of the reconstruction.

Okorokov AL, Waugh A, Hodgkinson J, Murthy A, Hong HK, Leo E, Sherman MB, Stoeber K, Orlova EV, Williams GH. Hexameric ring structure of human MCM10 DNA replication factor. (2007) EMBO Rep. 10, 925-30

EM group web page Elena Orlova