Conformational rearrangements of SV40 large T antigen during early replication events

Abstract

The Simian virus 40 (SV40) large tumor antigen (LTag) functions as the replicative helicase and initiator for viral DNA replication. For SV40 replication, the first essential step is the assembly of an LTag double hexamer at the origin DNA that will subsequently melt the origin DNA to initiate fork unwinding. In this study, we used three-dimensional cryo-electron microscopy to visualize early events in the activation of DNA replication in the SV40 model system. We obtained structures of wild-type double-hexamer complexes of LTag bound to SV40 origin DNA, to which atomic structures have been fitted. Wild-type LTag was observed in two distinct conformations: In one conformation, the central module containing the J-domains and the origin binding domains of both hexamers is a compact closed ring. In the other conformation, the central module is an open ring with a gap formed by rearrangement of the N-terminal regions of the two hexamers, potentially allowing for the passage of single-stranded DNA generated from the melted origin DNA. Double-hexamer complexes containing mutant LTag that lacks the N-terminal J-domain show the central module predominantly in the closed-ring state. Analyses of the LTag C-terminal regions reveal that the LTag hexamers bound to the A/T-rich tract origin of replication and early palindrome origin of replication elements are structurally distinct. Lastly, visualization of DNA density protruding from the LTag C-terminal domains suggests that oligomerization of the LTag complex takes place on double-stranded DNA.

Figure 1

Cryo-EM analysis of wt LTag and LTag108-627 double hexamers at the SV40 origin of replication. (a) Sequence scheme of wt LTag and LTag108-627. The N-terminal region is indicated with a red line. The plot indicates the intrinsic ordered (green) or disordered (red) nature of LTag sequences predicted by the server FoldIndex© . (b) Reference-free 2D averages of wt and mutant LTag Cryo-EM images. Red lines indicate the orientation of the central region of each hexamer (c) Cryo-EM maps of wtLTag-ori complex structures in the parallel and displaced conformations. Annotations indicate each of the two hexamers (Hex 1 and 2) and the C-terminal (C-ter) and central regions. (d) Cryo-EM map of the single class found for LTag108-627 (e) A predicted dsDNA structure of the SV40 ori as calculated by the web server bent.it© is superimposed to LTag108-627 map. The central perfect palindrome is depicted in green (P1, P4) and yellow (P2, P3) and the flanking regions in red (A/T-rich region) and in purple (EP region). Arrows point towards the edges of the flanking regions, where a marked bend is predicted.

Figure 2

Structure of the central region of LTag. (a) Top view of the fitting of the atomic structure of two OBD hexamers into LTag108-627 central region. The OBDs are named as in . The distances between domains b–e and c–f (~23-24Å) and between domains a–d (~35Å ) are represented by arrows. (b) Side view of the same fitting. Red dots indicate the location of each OBD N-terminus. The minor cathetus of the triangles depict the pitch of each OBD hexamer and the hypotenuse depicts the direction of the opening of the spiral, where is located the gap. A red square indicates the localization of the LTag108-627 central region. (c) Comparison of the parallel conformation of the wt LTag central region with the LTag108-627 central region. The left panel displays the wt central region in two colors. The green color represents the mass similar to the mutant structure and the red one, the extra mass in the wt map. The right panel shows the difference map between the central regions of the wt and mutant densities displayed as a red mesh on the mutant central domain structure.

Figure 3

Wt LTag C-terminal domain structures at the SV40 ori. (a) Diagram of LTag double hexamer assembled onto an asymmetric DNA probe, which extends beyond the LTag-occluded region at the AT end (blue). The probe used for wt LTag (dark blue) extends 7 nucleotides more than for LTag108-627 (light blue). The central perfect palindrome is depicted in yellow and the flanking regions in red (A/T-rich region) and in purple (EP region). (b) Raw images of LTag double hexamer complex. The hexamers with the protruding DNA have been oriented to the upper part of the image. (c) Class averages with a clear protruding DNA obtained by classification with a combination of MLF2D and KerDenSOM . (d) Maps of wt LTag C-terminal regions bound to A/T-rich (AT-Cter) and EP (EP-Cter) regions displayed at the same threshold and their difference map. Central slices of each map are shown as insets. In the difference maps, red mesh shows extra density in AT-Cter and green mesh, extra density in EP-Cter. Bars represent 10nm.

Figure 4

LTag108-627 C-terminal domain structures. (a) Maps of LTag108-627 C-terminal regions bound to SV40 ori. As in Figure 3, AT-Cter and EP-Cter are indicated. Central slices of each map are shown as insets. In the difference maps, red mesh shows extra density in AT-Cter and green mesh, extra density in EP-Cter. Bar represents 10nm (b) Side cut-away views of the ADP-helicase domain hexamer structure (yellow) fitted into LTag108-627 EP-Cter and AT-Cter maps. The β-hairpins implicated in DNA interaction are shown in cyan. (c) Difference map of wt LTag versus LTag108-627 AT-Cter domains. Difference densities (red mesh) are displayed onto a side view of the LTag108-627 AT-Cter fitting. The residue 627 of each LTag helicase domain is depicted as yellow spheres.

Figure 5

Model for the initiation mechanism of SV40 DNA replication. LTag double hexamer assembled onto ori sequence in the presence of ADP (a) and in the presence of ATP (b). The bubbles indicate the distortion of the DNA structure. The distortion of the A/T- rich region requires the presence of ATP and the presence of the region 121–135 , but not ATP hydrolysis ; ; . (c) The equilibrium between the straight and displaced conformations could be the mechanism for the ssDNA exit. (d) ATP hydrolysis powers the DNA unwinding.