The mechanism of DNA unwinding by the eukaryotic replicative helicase

• Published in: Nature communications 2019-05, Vol.10 (1), p.2159-2159, Article 2159
• Main Authors: Burnham, Daniel R., Kose, Hazal B., Hoyle, Rebecca B., Yardimci, Hasan
• Format: Article
• Language: English
• Subjects: 631/337/151; 631/337/2265; Cdc45 protein; Cell division; Deoxyribonucleic acid; DNA; DNA biosynthesis; DNA helicase; DNA Helicases - isolation & purification; DNA Helicases - metabolism; DNA Replication; Drosophila Proteins - isolation & purification; Drosophila Proteins - metabolism; Eukaryotes; Fruit flies; Genomes; Humanities and Social Sciences; Magnetic Phenomena; Models, Molecular; multidisciplinary; Optical Tweezers; Random walk; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Replication; Science; Science (multidisciplinary); Single Molecule Imaging - methods; Unwinding
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-09896-2

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. How the eukaryotic helicase unzips DNA during replication is not well understood. By measuring the real-time motion of purified CMG unwinding DNA with magnetic tweezers, the authors reveal the dynamics where isolated CMG unwinds via a biased random walk with proclivity to pause.