Ilic Stefan, Cohen Shira, Afek Ariel, Gordan Raluca, Lukatsky David B, Akabayov Barak
Department of Chemistry, Ben-Gurion University of the Negev.
Center for Genomic and Computational Biology, Department of Biostatistics and Bioinformatics, Duke University.
J Vis Exp. 2019 Oct 8(152). doi: 10.3791/59737.
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.
DNA引发酶合成短RNA引物，在DNA复制过程中，滞后链上的冈崎片段通过DNA聚合酶起始DNA合成。原核生物中类DnaG引发酶与DNA的结合发生在特定的三核苷酸识别序列处。这是冈崎片段形成过程中的关键步骤。用于确定DNA引发酶DNA识别序列的传统生化工具提供的信息有限。通过基于高通量微阵列的结合测定和连续的生化分析，已表明：1）特定的结合环境（识别位点的侧翼序列）会影响DNA引发酶与其模板DNA的结合强度；2）引发酶与DNA的更强结合会产生更长的RNA引物，表明该酶具有更高的持续合成能力。这种方法结合了蛋白质结合微阵列（PBM）和引发酶活性测定，被称为高通量引发酶分析（HTPP），它能够以前所未有的时间和规模对DNA引发酶的特定序列识别进行表征。
