Mechanism of Bidirectional Leading-Strand Synthesis Establishment at Eukaryotic DNA Replication Origins

Valentina Aria,Joseph T.P Yeeles

doi:10.1016/j.molcel.2018.10.019

Abstract

SummaryDNA replication commences at eukaryotic replication origins following assembly and activation of bidirectional CMG helicases. Once activated, CMG unwinds the parental DNA duplex and DNA polymerase α-primase initiates synthesis on both template strands. By utilizing an origin-dependent replication system using purified yeast proteins, we have mapped start sites for leading-strand replication. Synthesis is mostly initiated outside the origin sequence. Strikingly, rightward leading strands are primed left of the origin and vice versa. We show that each leading strand is established from a lagging-strand primer synthesized by the replisome on the opposite side of the origin. Preventing elongation of primers synthesized left of the origin blocked rightward leading strands, demonstrating that replisomes are interdependent for leading-strand synthesis establishment. The mechanism we reveal negates the need for dedicated leading-strand priming and necessitates a crucial role for the lagging-strand polymerase Pol δ in connecting the nascent leading strand with the advancing replisome.

Highlights

Bidirectional DNA replication is initiated from specific regions of the genome, termed origins
In vivo (Daigaku et al, 2015; Garbacz et al, 2018) and in vitro (Yeeles et al, 2017) experiments have indicated that, in addition to its role in lagging-strand synthesis, Pol d might participate in the initiation of leading-strand replication via a polymerase switch mechanism, with the 3ʹ end of the nascent leading strand sequentially transferred from Pol a to Pol d to CMGE. Why such an elaborate mechanism may be required is unknown, as is the frequency by which the two pathways are utilized. We have addressed these outstanding questions by mapping start sites for leading-strand replication at two S. cerevisiae replication origins using a reconstituted replication system (Taylor and Yeeles, 2018; Yeeles et al, 2015, 2017), determining the basis of Pol a recruitment to these sites, and defining the pathway by which the 3ʹ end of the nascent leading strand is connected to CMGE following primer synthesis
Previous work showed that Pol d is dispensable for leading-strand synthesis at replication forks reconstituted from purified S. cerevisiae proteins (Yeeles et al, 2015, 2017), which might indicate that pathway 1 is operative, omission of Pol d may not discriminate between the two pathways if free Pol ε is able to substitute in pathway 2, albeit less efficiently

Summary

Introduction

Bidirectional DNA replication is initiated from specific regions of the genome, termed origins. Assembly of the DNA replication machinery (replisome) begins in the G1 phase of the cell cycle when the ATP-dependent motor component of the replicative helicase, the hexameric Mcm complex (MCM), is loaded at origins by the origin recognition complex (ORC), Cdc and Cdt (Bell and Kaguni, 2013; Bell and Labib, 2016). CMG assembly and activation require multiple ‘‘firing factors’’ and are coupled to the initial untwisting and subsequent unwinding of duplex DNA at the replication origin (Douglas et al, 2018). Activated CMG translocates 3ʹ-5ʹ along the leading-strand template in an N-terminus-first orientation (Douglas et al, 2018; Georgescu et al, 2017; Moyer et al, 2006), and the two CMG complexes must pass one another before extensive template unwinding and DNA synthesis can occur

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Discussion

Conclusion