DNA Sequence Context Controls the Binding and Processivity of the T7 DNA Primase

Ariel Afek, Stefan Ilic, John Horton, David B Lukatsky, Raluca Gordan, Barak Akabayov

Research output: Contribution to journalArticlepeer-review


Primases are key enzymes involved in DNA replication. They act on single-stranded DNA and catalyze the synthesis of short RNA primers used by DNA polymerases. Here, we investigate the DNA binding and activity of the bacteriophage T7 primase using a new workflow called high-throughput primase profiling (HTPP). Using a unique combination of high-throughput binding assays and biochemical analyses, HTPP reveals a complex landscape of binding specificity and functional activity for the T7 primase, determined by sequences flanking the primase recognition site. We identified specific features, such as G/T-rich flanks, which increase primase-DNA binding up to 10-fold and, surprisingly, also increase the length of newly formed RNA (up to 3-fold). To our knowledge, variability in primer length has not been reported for this primase. We expect that applying HTPP to additional enzymes will reveal new insights into the effects of DNA sequence composition on the DNA recognition and functional activity of primases.
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•New HTPP workflow enables high-throughput profiling of primase binding and activity•Sequence context of GTC recognition sites strongly influences binding by T7 primase•Processivity of the T7 primase is significantly affected by template sequence•T7 primase forms longer primers from templates with higher DNA-binding affinity
Biochemical Mechanism; Molecular Biology; Molecular Genetics
Original languageEnglish
Pages (from-to)141-147
Number of pages7
Early online date27 Mar 2018
StatePublished - 27 Apr 2018


  • Biochemical Mechanism
  • Molecular Biology
  • Molecular Genetics

All Science Journal Classification (ASJC) codes

  • General


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