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

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

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere103367
JournalEMBO Journal
DOIs
StateAccepted/In press - Jan 1 2020

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Keywords

  • DNA polymerase
  • exonuclease activity
  • primer shuttling
  • replication hurdles
  • translocation

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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