Ryzhenkova, K. and Coscia, F. and Chaban, Yuriy and Sanders, C. and Orlova, Elena (2016) Structural study of the BPV E1 helicase/DNA complex using electron microscopy. In: UNSPECIFIED (ed.) European Microscopy Congress 2016 Proceedings. Wiley-VCH Verlag GmbH & Co. KGaA, pp. 39-40. ISBN 9783527342976.
Abstract
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 cell‐free 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 three‐dimensional (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), full‐length E1 (E1FL), a single‐labelled E1FL‐replication fork DNA complex with monovalent tetrameric streptavidin (MTS) on the dsDNA of the fork and a double‐labelled 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 6‐fold symmetry and without applying any symmetry. EM demonstrated that the E1FL helicase forms a hexamer that has a diameter of 130Å and a height of 100Å, 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 12‐residue 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 5′ 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 DIG‐FAB to reveal the 5′ 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 N‐terminus and oriDNA‐binding domain (OBD), and the 5′ ssDNA exits the molecule between the collar domain and OBD. The angle between the point of dsDNA entrance and 5′ ssDNA exit was ~170°. Prior to our research, the most accepted model of “steric exclusion” for dsDNA unwinding suggested that the active 3′ 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 5′ 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.
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/23579 |
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