Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles.

Anupam Singh,Y Whitney Yin,Kevin D Raney,Divya Nandakumar,Smita S Patel,Manjula Pandey

doi:10.15252/embj.2019103367

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.

Highlights

Replicative DNA polymerases (DNAPs) are responsible for faithfully copying the genomic DNA
The 70 bp minicircle DNA contains a priming sequence for the T7 primase that makes short RNA primers in the presence of ATP and CTP, which are elongated by T7 DNAP to make the Okazaki lagging strand DNA products
What type of translocation problems occurs during leading strand synthesis? We have shown that T7 DNAP can unwind up to two base-pairs of the duplex DNA at the fork junction (Nandakumar et al, 2015)

Summary

Introduction

Replicative DNA polymerases (DNAPs) are responsible for faithfully copying the genomic DNA. These polymerases have a high selectivity for the correct incoming nucleotide, which keeps replication error rates low between 10À4 and 10À6 (Kunkel & Bebenek, 2000; Alba, 2001; Kunkel, 2004; Johansson & Dixon, 2013). Exo-site mutants of replicative Pol e and Pol d DNAPs are found in tumors, and many of them show an ultramutational phenotype (Albertson et al, 2009). The ultramutational phenotype is not explained entirely by the loss of proofreading activity (Kane & Shcherbakova, 2014; Li et al, 2018), suggesting that there are additional roles of the exonuclease activity that prevent mutagenicity

Methods

Results

Conclusion