JoVE Logo

Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Abstract

Biochemistry

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

Published: October 8th, 2019

DOI:

10.3791/59737

1Department of Chemistry, Ben-Gurion University of the Negev, 2Center for Genomic and Computational Biology, Department of Biostatistics and Bioinformatics, Duke University

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. 

Explore More Videos

Keywords DNA Primase

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved