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

摘要

The proofreading exonuclease activity of replicative polymerase excises misincorporated nucleotides during 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 polymerases establish its generality. We show that excessive excision of correctly incorporated nucleotides is not due to events such as processive degradation of nascent 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 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.