The mechanism of DNA unwinding by the eukaryotic replicative helicase

Daniel R Burnham,Hasan Yardimci,Hazal B Kose,Rebecca B Hoyle

doi:10.1038/s41467-019-09896-2

Daniel R Burnham, Hasan Yardimci + Show 2 more

Open Access

https://doi.org/10.1038/s41467-019-09896-2

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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.

Highlights

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome
The substrate was incubated with DmCMG in the presence of ATPγS and unwinding initiated by addition of ATP
~4–5 chamber volumes of temperature-equilibrated CMG-free running buffer, containing ATP, is drawn through the chamber (Fig. 1d). This ensures only previously loaded CMG remains in the sample chamber, preventing multiple helicases acting on single DNA substrates

Summary

Introduction

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+ heterohexameric complex, Mcm[2,3,4,5,6,7], together with GINS and Cdc[45] form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG) It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. To explain our data we build a quantitative kinetic model to underpin a biophysical description of the unwinding mechanism and establish that CMG unwinds DNA via a biased random walk with propensity to pause. Without this understanding of the foundation of the eukaryotic replisome, further insight into how replication dysfunction can be a major source of genome instability will be slowed

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