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

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. Synopsis Replication hurdles during DNA synthesis promote frequent shuttling of the primer end from the polymerase to the exonuclease site, resulting in excessive excision of correctly incorporated nucleotides and possibly serving as a proofreading mechanism protecting primer ends in the exonuclease site from mutagenic extension. Excessive excision of correctly incorporated nucleotides occurs in diverse replicative DNA polymerases during leading‐ and lagging‐strand synthesis due to generation of polymerase‐inhibited states. Excessive excision results from frequent shuttling of the primer end from the polymerase site to the exonuclease site. DNA secondary structures drive excision of correctly incorporated nucleotides during lagging‐strand synthesis. Polymerase‐helicase uncoupling drives excision during leading strand synthesis. Graphical Abstract Frequent shuttling of primer ends between polymerase‐ and exonuclease‐active sites may serve as proofreading mechanism preventing them from mutagenic extension.