Excessive Excision of Correct Nucleotides During DNA Synthesis Explained by Replication Hurdles

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 2020 Feb 11

PMID 32037587

Citations 12

Authors

Anupam Singh

Manjula Pandey

Divya Nandakumar

Kevin D Raney

Y Whitney Yin

Smita S Patel

Affiliations

Soon will be listed here.

Abstract

The proofreading exonuclease activity of replicative DNA polymerase excises misincorporated nucleotides during DNA synthesis, but these events are rare. Therefore, we were surprised to find that T7 replisome excised nearly 7% of correctly incorporated nucleotides during leading and lagging strand syntheses. Similar observations with two other DNA polymerases establish its generality. We show that excessive excision of correctly incorporated nucleotides is not due to events such as processive degradation of nascent DNA or spontaneous partitioning of primer-end to the exonuclease site as a "cost of proofreading". Instead, we show that replication hurdles, including secondary structures in template, slowed helicase, or uncoupled helicase-polymerase, increase DNA reannealing and polymerase backtracking, and generate frayed primer-ends that are shuttled to the exonuclease site and excised efficiently. Our studies indicate that active-site shuttling occurs at a high frequency, and we propose that it serves as a proofreading mechanism to protect primer-ends from mutagenic extensions.

Citing Articles

Torsion is a Dynamic Regulator of DNA Replication Stalling and Reactivation.

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Reclassification of family A DNA polymerases reveals novel functional subfamilies and distinctive structural features.

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Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB.

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