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The mechanism of DNA unwinding by the eukaryotic replicative helicase

The mechanism of DNA unwinding by the eukaryotic replicative helicase
The mechanism of DNA unwinding by the eukaryotic replicative helicase
Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.
2041-1723
Burnham, Daniel
af899583-ded1-47d0-a931-466acf405411
Kose, Hazal
613a71db-ac1a-4f3e-8557-692de422c92d
Hoyle, Rebecca
e980d6a8-b750-491b-be13-84d695f8b8a1
Yardimci, Hasan
667ea63a-c4b3-4dde-bd22-b4f17c5386d9
Burnham, Daniel
af899583-ded1-47d0-a931-466acf405411
Kose, Hazal
613a71db-ac1a-4f3e-8557-692de422c92d
Hoyle, Rebecca
e980d6a8-b750-491b-be13-84d695f8b8a1
Yardimci, Hasan
667ea63a-c4b3-4dde-bd22-b4f17c5386d9

Burnham, Daniel, Kose, Hazal, Hoyle, Rebecca and Yardimci, Hasan (2019) The mechanism of DNA unwinding by the eukaryotic replicative helicase. Nature Communications, 10 (1), [2159]. (doi:10.1038/s41467-019-09896-2).

Record type: Article

Abstract

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.

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More information

Accepted/In Press date: 5 April 2019
e-pub ahead of print date: 14 May 2019

Identifiers

Local EPrints ID: 430815
URI: http://eprints.soton.ac.uk/id/eprint/430815
ISSN: 2041-1723
PURE UUID: 277c1abb-cb87-440d-874e-b7317e943228
ORCID for Rebecca Hoyle: ORCID iD orcid.org/0000-0002-1645-1071

Catalogue record

Date deposited: 14 May 2019 16:30
Last modified: 26 Nov 2021 02:56

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Contributors

Author: Daniel Burnham
Author: Hazal Kose
Author: Rebecca Hoyle ORCID iD
Author: Hasan Yardimci

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