Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model

Hazal B Kose,Nicolai B Larsen,Julien P Duxin,Hasan Yardimci

doi:10.1016/j.celrep.2019.01.086

Hazal B Kose, Nicolai B Larsen + Show 2 more

Open Access

https://doi.org/10.1016/j.celrep.2019.01.086

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Abstract

SummaryProgression of DNA replication depends on the ability of the replisome complex to overcome nucleoprotein barriers. During eukaryotic replication, the CMG helicase translocates along the leading-strand template and unwinds the DNA double helix. While proteins bound to the leading-strand template efficiently block the helicase, the impact of lagging-strand protein obstacles on helicase translocation and replisome progression remains controversial. Here, we show that CMG and replisome progressions are impaired when proteins crosslinked to the lagging-strand template enhance the stability of duplex DNA. In contrast, proteins that exclusively interact with the lagging-strand template influence neither the translocation of isolated CMG nor replisome progression in Xenopus egg extracts. Our data imply that CMG completely excludes the lagging-strand template from the helicase central channel while unwinding DNA at the replication fork, which clarifies how two CMG helicases could freely cross one another during replication initiation and termination.

Highlights

We show that proteins that exclusively interact with the lagging-strand template do not impact CMG or replisome progression, supporting strand exclusion as the preferred mechanism of DNA unwinding by CMG
Biotin–Streptavidin Complex on the Leading-Strand Template Inhibits DNA Unwinding by CMG To understand how CMG interacts with DNA at the replication fork, we purified Drosophila melanogaster CMG (DmCMG) by overexpressing all 11 subunits of the complex in insect cells (Figure S1A) (Ilves et al, 2010)
A fork DNA substrate bearing 40-nucleotide poly-T sequence on both single-stranded DNA tails can bind two CMG complexes, replacing the 50 lagging-strand arm with repeats of a GGCA sequence leads to only one CMG binding, as this sequence forms secondary hairpin-like structures preventing CMG assembly on the 50 tail (Petojevic et al, 2015)

Summary

Introduction

Many origin sites on DNA are ‘‘licensed’’ (Blow and Laskey, 1988; Chong et al, 1995; Kubota et al, 1995; Madine et al, 1995) for replication in the Gap 1 (G1) phase by the loading of hetero-hexameric Minichromosome maintenance (Mcm) 2-7 complexes through the collective actions of Origin Recognition Complex (ORC), Cdt, and Cdc (Coster and Diffley, 2017; Coster et al, 2014; Evrin et al, 2009; Kang et al, 2014; Remus et al, 2009; Ticau et al, 2015). Mcm complexes, which assemble around double-stranded DNA (dsDNA) as double hexamers, are called pre-replication complexes (preRCs). PreRCs remain inactive until cells enter into the Synthesis (S) phase, after which two protein kinases, namely, Cyclin-Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK), are involved in activating the helicase. Mcm for the recruitment of Cdc and the GINS complex (Douglas et al, 2018; Heller et al, 2011; Labib, 2010; Muramatsu et al, 2010; Sheu and Stillman, 2006; Tanaka et al, 2007; Zegerman and Diffley, 2007). Recruitment of a number of other factors establishes the replisome complex, and the unwound DNA is replicated through the synthesis of the leading and lagging strands by polymerase epsilon and delta, respectively (Lujan et al, 2016)

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