Molecular architecture of the human GINS complex

Abstract

Chromosomal DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of these is the GINS complex, which is required for initiation and elongation phases in eukaryotic DNA replication. The GINS complex consists of four paralogous subunits. At the G1/S transition, GINS is recruited to the origins of replication where it assembles with cell-division cycle protein (Cdc)45 and the minichromosome maintenance mutant (MCM)2-7 to form the Cdc45/Mcm2-7/GINS (CMG) complex, the presumed replicative helicase. We isolated the human GINS complex and have shown that it can bind to DNA. By using single-particle electron microscopy and three-dimensional reconstruction, we obtained a medium-resolution volume of the human GINS complex, which shows a horseshoe shape. Analysis of the protein interactions using mass spectrometry and monoclonal antibody mapping shows the subunit organization within the GINS complex. The structure and DNA-binding data suggest how GINS could interact with DNA and also its possible role in the CMG helicase complex.

Figure 1

The human GINS complex is a heterotetramer that binds to DNA. (A) SDS–polyacrylamide gel electrophoresis of the purified recombinant human GINS (hGINS) complex shows four bands and their identity as human GINS subunits was confirmed by using mass spectrometry. (B) Nanoelectrospray mass spectrometric analysis of the intact human GINS complex shows a well-resolved charge state series (labelled 23+ to 18+), which is consistent with the presence of the four subunits in stoichiometric amounts. Inset: gas-phase acceleration of the isolation at approximately 4.700 m/z (21+ charge state) and tandem mass spectrometry clearly showed the dissociation of the subunit Psf2 from the intact complex. (C) Scheme of the DNA probes forming different structures used for EMSA. (D) EMSA of the human GINS complex. Increasing amounts of the human GINS complex were used, whereas the probe concentration was constant. EMSA, electrophoretic mobility shift assays.

Figure 2

Electron microscopy and three-dimensional structure of the human GINS complex. (A) Representative area of the human GINS micrographs. Some images of human GINS single molecules are indicated by asterisks. (B) Gallery of single particles showing some representative views. (C) A collection of selected projections of the final volume (Proj) and three-dimensional averages of the images within the corresponding class (Aver). (D–F) Different views of the reconstructed volume from human GINS.

Figure 3

Identification of human GINS subcomplexes by mass spectrometry. Spectrum from (A) mass spectrometry and (B) tandem mass spectrometry analysis of the human GINS complex after disruption of the complex using 42% methanol. Two charge state series were observed in the m/z region 2,800–4,250 (A, inset). The measured masses (47,758 Da, light green squares; 70,895 Da, magenta stars) indicate that the two series correspond to the Psf2 and Sld5 dimer, and the heterotrimer composed of Psf1, Psf2 and Sld5. (B) The dissociation products Psf2 (green diamonds) and Sld5 (pink circles) of the 16+ charge state (highlighted in red) of the Psf2, Sld5 dimer confirm its composition. (C) Interactions of the GINS subunits.

Figure 4

Localization of the Psf2 in the human GINS–Fab complex. (A) Electron microscopic field of a negatively stained sample of purified human GINS–Fab complex. Some particles are indicated with asterisks. (B) Panel containing projections (Proj) and their corresponding class averages (Aver) obtained after refinement. (C) Top and (D) front views of the three-dimensional structure of human GINS–Fab complex and (E,F) the fitting of human GINS and atomic structure of Fab (2HFF.pdb) coloured in blue and magenta, respectively.

Figure 5

Hypothetical models of the function of Cdc45/Mcm2–7/GINS complex function (see Discussion for details). (A) The MCM2–7 complex (yellow) interacts with double-stranded DNA (pink and magenta), and GINS (blue) could act as a plough preventing the separated strands from the unwound DNA from re-associating. (B) In this model the MCM2–7 interacts with single-stranded DNA, and the GINS complex keeps the single strand available to other replication factors. Cdc45, cell-division cycle 45; MCM2–7, minichromosome maintenance mutant 2–7.