Hesketh, E.L. and Parker-Manuel, R.P. and Chaban, Y. and Satti, R. and Coverley, D. and Orlova, Elena and Chong, James P.J. (2015) DNA induces conformational changes in a recombinant human minichromosome maintenance complex. Journal of Biological Chemistry , jbc.M114.622738. ISSN 0021-9258.
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Abstract
ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has only been observed in the context of a co-purified Cdc45-MCM-GINS (CMG) complex. Here we describe the production of recombinant human MCM complex (hMCM) in E. coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single particle asymmetric electron microscopy reconstruction, we demonstrate recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for the biochemical reconstitution of the human replicative helicase.
Metadata
| Item Type: | Article |
|---|---|
| Keyword(s) / Subject(s): | ATPase, DNA helicase, DNA replication, electron microscopy (EM), recombinant protein expression, MCM, minichromosome maintinence |
| School: | Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences |
| Research Centres and Institutes: | Structural Molecular Biology, Institute of (ISMB) |
| Depositing User: | Administrator |
| Date Deposited: | 12 Feb 2015 13:54 |
| Last Modified: | 02 Aug 2023 17:15 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/11599 |
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