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

doi:10.1016/j.isci.2018.03.019

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

iScience. 2018 Apr 27:2:141-147. doi: 10.1016/j.isci.2018.03.019. Epub 2018 Mar 27.

Authors

Ariel Afek¹, Stefan Ilic², John Horton¹, David B Lukatsky³, Raluca Gordan⁴, Barak Akabayov⁵

Affiliations

¹ Center for Genomic and Computational Biology, Department of Biostatistics and Bioinformatics, Duke University, Durham, NC 27708, USA.
² Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
³ Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Electronic address: lukatsky@bgu.ac.il.
⁴ Center for Genomic and Computational Biology, Department of Biostatistics and Bioinformatics, Duke University, Durham, NC 27708, USA; Department of Computer Science, Department of Molecular Genetics and Microbiology, Duke University, Durham, NC 27708, USA. Electronic address: raluca.gordan@duke.edu.
⁵ Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Electronic address: akabayov@bgu.ac.il.

Abstract

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.

Keywords: Biochemical Mechanism; Molecular Biology; Molecular Genetics.