Roles for DNA polymerase \u03b4 in initiating and terminating leading strand DNA replication

Zhi-Xiong Zhou,Thomas A Kunkel,Marta A Garbacz,Scott A Lujan,Adam B Burkholder

doi:10.1038/s41467-019-11995-z

Zhi-Xiong Zhou, Thomas A Kunkel + Show 3 more

Open Access

https://doi.org/10.1038/s41467-019-11995-z

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Abstract

Most current evidence indicates that DNA polymerases ε and δ, respectively, perform the bulk of leading and lagging strand replication of the eukaryotic nuclear genome. Given that ribonucleotide and mismatch incorporation rates by these replicases influence somatic and germline patterns of variation, it is important to understand the details and exceptions to this overall division of labor. Using an improved method to map where these replicases incorporate ribonucleotides during replication, here we present evidence that DNA polymerase δ universally participates in initiating leading strand synthesis and that nascent leading strand synthesis switches from Pol ε to Pol δ during replication termination. Ribonucleotide maps from both the budding and fission yeast reveal conservation of these processes. These observations of replisome dynamics provide important insight into the mechanisms of eukaryotic replication and genome maintenance.

Highlights

Most current evidence indicates that DNA polymerases ε and δ, respectively, perform the bulk of leading and lagging strand replication of the eukaryotic nuclear genome
Averaged across efficient replication origins, we recently showed that during initiation of leading strand synthesis, about 180 bp, or roughly one Okazaki fragment, is synthesized by Pol δ between initial priming by polymerase α (Pol α)-primase and extensive synthesis by Pol ε14
The main changes are switching from alkaline hydrolysis to E. coli RNase HII digestion, which cleaves at single embedded ribonucleotides, restriction digestion to introduce internal standards, inclusion of non-treatment control and 5′ end blocking by phosphatase

Summary

Introduction

Most current evidence indicates that DNA polymerases ε and δ, respectively, perform the bulk of leading and lagging strand replication of the eukaryotic nuclear genome. Using an improved method to map where these replicases incorporate ribonucleotides during replication, here we present evidence that DNA polymerase δ universally participates in initiating leading strand synthesis and that nascent leading strand synthesis switches from Pol ε to Pol δ during replication termination Ribonucleotide maps from both the budding and fission yeast reveal conservation of these processes. Studies of unchallenged DNA replication in yeast systems, using mutations and genomic ribonucleotides as biomarkers of polymerase activity, indicate that Pol δ conducts the majority of discontinuous lagging strand Okazaki fragment synthesis, while Pol ε accomplishes the majority of continuous leading strand DNA replication[1,2,3,4] This canonical model of a Replisome elements & canonical divison of labor. We show prevalent exceptions to the canonical polymerase division of labor at origins, and we suggest an unexpected mechanism of replication termination in eukaryotes

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