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. 2017 Jan 10;7:40188.
doi: 10.1038/srep40188.

New Insights Into the GINS Complex Explain the Controversy Between Existing Structural Models

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Free PMC article

New Insights Into the GINS Complex Explain the Controversy Between Existing Structural Models

Marta Carroni et al. Sci Rep. .
Free PMC article

Abstract

GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron microscopy (EM), we show that GINS is a compact tetramer in solution as observed in crystal structures, but also forms a double-tetrameric population, detectable by EM. This may represent an intermediate step towards the assembly of two replicative helicase complexes at origins, moving in opposite directions within the replication bubble. Reconstruction of the double-tetrameric form, combined with small-angle X-ray scattering data, allows the localisation of the B domain of the Psf1 subunit in the free GINS complex, which was not visible in previous studies and is essential for the formation of a functional replication fork.

Figures

Figure 1
Figure 1. Structure and architecture of GINS complex.
(a) Ribbon representation of the crystallographic structure of human GINS (PDB ID: 2EHO) (b) Schematic diagram of the GINS subunits, highlighting the domain swap between the α-helical (A) and the β-rich (B) domains. (c) EM map of hGINS (EMD-1355) showing a C-shaped molecule. (d) To resolve the discrepancy between crystallographic and EM data a large rearrangement of the four GINS subunits has been proposed.
Figure 2
Figure 2. Dimerisation of hGINS.
(a) Size-exclusion chromatography profile of a monomeric peak fraction run minutes (dashed line) or days (solid line) after elution. In the inset is indicated the fraction that was run again (labelled 1 in red) and the corresponding Coomassie-stained SDS gel of the sample. Coomassie-stained 12% SDS-polyacrylamide gel show the four subunits eluting from the peaks. (b) SEC-MALLS analysis of hGINS complex. Elution profiles generated by applying 100 μL of 5 (black line) and 10 mg/mL (red line) hGINS onto a Superdex 200 column coupled with differential refractive index detection are shown. Arrows indicate the MW measurements of the species eluted in the two peaks. (c) Samples from the two size exclusion chromatography peaks were stained and visualised by EM. Scale bar is 200 Å.
Figure 3
Figure 3. Three dimensional reconstruction of the hGINS double tetramer.
(a) Outline of the 3D reconstruction procedure. Eigen-images indicate the presence of 2-fold symmetry. (b) Double tetramer EM map with a fitted crystallographic dimer (PDB ID: 2EHO, chains E to L). The green asterisks indicate the location of the last residue of Psf1.
Figure 4
Figure 4. SAXS analysis of the full-length hGINS complex.
(a) Experimental SAXS profile (blue crosses) and GNOM fit (yellow line). (b) Final model reconstructed from the scattering curve. The crystal structure of a GINS tetramer (PDB ID: 2EHO) was fitted onto the SAXS map; to illustrate the putative position of the Psf1 B domain, the equivalent region from Sld5 from has been fitted into the protrusion (in green). (c) The simulated SAXS curves from the crystallographic GINS structure (red), from a model including the exposed Psf1 B domain (green) and from the full-length GINS complex derived from the 4.7 Å EM MAP (PDB ID: 3JC5, magenta), are compared with the experimental SAXS data. An inset shows the fit at low angles. (d) Agreement between SAXS and EM. Two SAXS envelope (in grey and yellow, respectively) were overlapped with the EM double tetramer and compared with the EM double tetrameric map.

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