7 research outputs found
Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase
Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous. Such diversity has hindered identification of known or expected UDG-inhibitory activities in other genomes. The structural basis for UDG inhibition by ugi is well characterized; yet, paradoxically, the structure of the unbound p56 protein is enigmatically unrevealing of its mechanism. To resolve this conundrum, we determined the structure of a p56 dimer bound to UDG. A helix from one of the subunits of p56 occupies the UDG DNA-binding cleft, whereas the dimer interface forms a hydrophobic box to trap a mechanistically important UDG residue. Surprisingly, these p56 inhibitory elements are unexpectedly analogous to features used by ugi despite profound architectural disparity. Contacts from B-DNA to UDG are mimicked by residues of the p56 helix, echoing the role of ugiās inhibitory beta strand. Using mutagenesis, we propose that DNA mimicry by p56 is a targeting and specificity mechanism supporting tight inhibition via hydrophobic sequestration
Structural study of the BPV E1 helicase/DNA complex using electron microscopy
DNA replication is a key cellular process and is the basis for cell division. In order to understand replication initiation in mammalian cells, viral systems such as bovine papillomavirus (BPV) are used as simplified models of the process. BPV uses a single protein E1 to perform both DNA binding and unwinding functions essential for the initiation of replication (Figure A). Biochemical assays for a cellāfree replication initiation system has been established for BPV. To have a clearer understanding of a biological process at a molecular level, it is essential to determine the threeādimensional (3D) arrangement and dynamics of molecules and how macromolecular machines are assembled. Electron microscopy (EM), single particle analysis and image processing have been used in this work to reveal the structures of the BPV E1 helicase domain (E1HD), fullālength E1 (E1FL), a singleālabelled E1FLāreplication fork DNA complex with monovalent tetrameric streptavidin (MTS) on the dsDNA of the fork and a doubleālabelled E1FLwith MTS on the dsDNA and FAB on the ssDNA of the fork.
The structure of BPV E1FL in complex with DNA was initially determined by EM both by applying 6āfold symmetry and without applying any symmetry. EM demonstrated that the E1FL helicase forms a hexamer that has a diameter of 130Ć
and a height of 100Ć
, consistent with an overall mass of ~410 kDa. The oligomer has a central channel inside the molecule, with a variable diameter. E1FL is a single polypeptide chain, and the OBD domain can be fitted into the structure in two different ways. In one case the DNA binding site would be located on the inner surface and in the other case it would be on the outer surface of the complex. To determine the orientation of the OBD domain, a 12āresidue epitope sequence GGYPYDVPDYAG was inserted into the OBD domain after the residue 226. EM has demonstrated that the antibody was bound to the E1FL. That proves that the binding site for AB is located on the outer surface of E1FL (Figure B). The points of the entrance of dsDNA and exit of 5ā² ssDNA were also determined in the complex of E1FL and DNA. Labelling with streptavidin was performed to reveal the position of the dsDNA, whereas ssDNA was labelled with DIGāFAB to reveal the 5ā² ssDNA position in an asymmetrical structure of the BPV E1 hexamer bound to a replication fork DNA substrate. Comparison of the 3D structures showed that dsDNA enters the molecule between the Nāterminus and oriDNAābinding domain (OBD), and the 5ā² ssDNA exits the molecule between the collar domain and OBD. The angle between the point of dsDNA entrance and 5ā² ssDNA exit was ~170Ā°.
Prior to our research, the most accepted model of āsteric exclusionā for dsDNA unwinding suggested that the active 3ā² ssDNA strand is pulled through the helicase motor and dsDNA is wedged apart outside the protein assembly (Figure C). Our structural observations indicated that strand separation is taking place inside E1 in a chamber above the helicase domain and that the 5ā² passive ssDNA strand leaves the assembly through a channel located on the opposite side to dsDNA entry. Therefore, our data suggest an alternative model for DNA unwinding by this general class of replication enzymes
Single particle EM study of the E1 helicase from Papillomavirus in complex with DNA
The papillomaviruses (PV) are small dsDNA tumour viruses of significant medical importance and the prototype of the group is bovine PV (BPVā1). PVs encode one highly conserved replication enzyme, E1, that acts as an initiator and a helicase. E1 forms hexamers and unwinds double stranded DNA (dsDNA) into single stranded DNA (ssDNA) products using the energy of ATP hydrolysis. However, how the helicase engages the replication fork at the molecular level is unclear. BPVā1 E1 is comprised of four domains: a regulatory domain (residues 1ā158), a sequence specific ori DNA binding domain (OBD, residues 159ā299) and the Cāterminal half (residues 300ā605, E1HD) that has helicase activity and is further divided into the oligomerisation domain (OD, residues 308ā378) and the AAA+ ATPase domain. A Cāterminal acidic tail domain (AT, residues 579ā605) is required for helicase processivity (Figure A). We have obtained structures of the full length E1 helicase (E1FL) in complex with a DNA replication fork with and without DNA labelling with protein tags. The 5ā end of dsDNA and the 5ā ssDNA end of the fork were labelled and helicase structures studied using single particle electron microscopy.
Negatively stained images (with 2% uranyl acetate) of E1FL/DNAfork/Fab /Streptavidin were taken and the structure obtained at a resolution of~ 20 Ć
(Figure B). We are currently working on the native structure by cryoEM (Figure C). The 3D reconstruction of E1FL (Figure D) has a threeātier organisation with wellādefined domains (Nāterminal, DBD, OD, and AAA+). Domains were localised by docking of available atomic structures. The 3D structure of the E1/ labelled DNA fork confirmed the triple ring organisation with an internal small chamber above the helicase motor domain where DNA unwinding appears to take place. Interestingly, dsDNA enters into the chamber via a side tunnel above the helicase motor domain, with the 5ĢāssDNA strand leaving through a narrow tunnel located on the opposite side, while the 3ĢāssDNA is pulled through the hexamer's central tunnel (Figure D). Our findings are confirmed by DNA footprint experiments and FRET experiments. Recently we analysed a cryoEM structure of the E1FL in complex with a DNA fork at subnanometer resolution that reveals the same structural organization and provides more detail on the interaction with DNA.
Our structural studies demonstrate that the process of DNA separation takes place inside the E1FL complex rather than on the exterior surface of E1. In the light of our results we suggest that the current āsteric exclusionā model for strand separation should be revised. A molecular understanding of E1 function will be essential to shed light on the early phase of DNA replication and will assist in the understanding of E1 as a therapeutic target of viral DNA replication