Coscia, F. and Ryzhenkova, K. and Chaban, Yuriy and Sanders, C. and Orlova, Elena (2016) Single particle EM study of the E1 helicase from Papillomavirus in complex with DNA. In: UNSPECIFIED (ed.) European Microscopy Congress 2016 Proceedings. Wiley-VCH Verlag GmbH & Co. KGaA, pp. 37-38. ISBN 9783527342976.
Abstract
The papillomaviruses (PV) are small dsDNA tumour viruses of significant medical importance and the prototype of the group is bovine PV (BPV‐1). 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. BPV‐1 E1 is comprised of four domains: a regulatory domain (residues 1‐158), a sequence specific ori DNA binding domain (OBD, residues 159‐299) and the C‐terminal half (residues 300‐605, E1HD) that has helicase activity and is further divided into the oligomerisation domain (OD, residues 308‐378) and the AAA+ ATPase domain. A C‐terminal acidic tail domain (AT, residues 579‐605) 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 5’ end of dsDNA and the 5’ 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 Å (Figure B). We are currently working on the native structure by cryoEM (Figure C). The 3D reconstruction of E1FL (Figure D) has a three‐tier organisation with well‐defined domains (N‐terminal, 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 5́‐ssDNA strand leaving through a narrow tunnel located on the opposite side, while the 3́‐ssDNA 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 ‘steric exclusion’ 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.
Metadata
| Item Type: | Book Section |
|---|---|
| School: | Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences |
| SWORD Depositor: | Mr Joe Tenant |
| Depositing User: | Mr Joe Tenant |
| Date Deposited: | 06 Sep 2018 08:10 |
| Last Modified: | 02 Aug 2023 17:43 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/23585 |
Statistics
Additional statistics are available via IRStats2.
Archive Staff Only (login required)
