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

EMBO J. 2020 Mar 16;39(6):e103367. doi: 10.15252/embj.2019103367. Epub 2020 Feb 9.


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.

Keywords: DNA polymerase; exonuclease activity; primer shuttling; replication hurdles; translocation.

MeSH terms

  • Bacteriophage T7 / enzymology
  • Bacteriophage T7 / genetics*
  • Catalytic Domain
  • DNA / biosynthesis*
  • DNA Primase / genetics
  • DNA Primase / metabolism*
  • DNA Primers / genetics
  • DNA Repair / genetics*
  • DNA Replication / genetics*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism
  • Mutation
  • Nucleotides / genetics


  • DNA Primers
  • Nucleotides
  • DNA
  • DNA Primase
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • gene b exonuclease