Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome

Max E Douglas,Andrew Purkiss,Julia Locke,Panchali Goswami,Patrik Eickhoff,John F X Diffley,Anne Early,Agnieszka Janska,Alessandro Costa,Ferdos Abid Ali,Andrea Nans,Alan M C Cheung

doi:10.1038/s41467-018-07417-1

Abstract

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.

Highlights

Eukaryotic origin firing depends on assembly of the Cdc45-maintenance protein complex (MCM)-GINS (CMG) helicase
To understand the role of the essential Pol epsilon modules during DNA replication, we studied the structure of a truncation mutant of the yeast tetrameric complex, lacking the dispensable, flexibly tethered N-terminal catalytic domain of Pol[228]
Given the relatively small size of our protein target and inherent flexibility detected in preliminary cryo-Electron microscopy (EM) characterization[28], we acquired a dataset at low defocus and using the Volta phase plate (VPP)

Summary

Introduction

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol[2], Dpb[2], Dpb[3], Dpb[4]. Reconstitution studies showed that the leading-strand polymerase Pol epsilon forms a stable complex with the CMG21,27, by binding to the ATPase tier of MCM via a non-catalytic domain[28]. Hetero-tetrameric Pol epsilon plays a key role in replisome maturation and origin activation[19,29,30] In this protein assembly, Dpb[3] and Dpb[4] are ancillary, DNA-binding subunits containing a histone fold[31]. It is clear that non-catalytic modules in Pol epsilon are required for helicase activation and origin firing[13,35,38], the molecular basis is poorly understood What role these inactivated domains play during fork progression is unclear[1]. Our data provide important insights into the distinct roles of Pol epsilon during DNA replication

Methods

Results

Conclusion