DNA sequence recognition by DNA primase using high-throughput primase profiling

Stefan Ilic, Shira Cohen, Ariel Afek, Raluca Gordan, David B. Lukatsky, Barak Akabayov

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.

Original languageEnglish
Article numbere59737
JournalJournal of Visualized Experiments
Volume2019
Issue number152
DOIs
StatePublished - 1 Oct 2019

Keywords

  • Biochemistry
  • DNA primase
  • DNA replication
  • DNA-binding
  • HTPP
  • High throughput primase profiling
  • Issue 152
  • Molecular biology
  • PBM
  • Protein binding microarray
  • Protein-DNA interaction
  • RNA primers

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