DNA Induces Conformational Changes in a Recombinant Human Minichromosome Maintenance Complex

Emma L Hesketh, Richard P Parker-Manuel, Yuriy Chaban, Rabab Satti, Dawn Coverley, Elena V Orlova, James P J Chong

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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.

Original languageEnglish
Article number622738
JournalThe Journal of biological chemistry
Publication statusPublished - 3 Feb 2015

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© 2015, The American Society for Biochemistry and Molecular Biology, Inc. This is an Open Access article made available under a Creative Commons Attribution licence which allows sharing and reuse provided that the authors and source are properly acknowledged.

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