Analysis of replicative polymerase usage by ribonucleotide incorporation

Andrea Keszthelyi, Izumi Miyabe, Katie Ptasińska, Yasukazu Daigaku, Karel Naiman, Antony M. Carr

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Mapping the usage of replicative DNA polymerases has previously proved to be technically challenging. By exploiting mutant polymerases that incorporate ribonucleotides into the DNA with a significantly higher proficiency than their wild-type counterparts, we and others have developed methods that can identify what proportion of each DNA strand (i.e., the Watson and Crick strands) is replicated by a specific DNA polymerase. The incorporation of excess ribonucleotides by a mutated polymerase effectively marks, in each individual cells, the DNA strand that is replicated by that specific mutated polymerase. Changes to DNA polymerase usage can be examined at specific loci by Southern blot analysis while a global analysis of polymerase usage can be achieved by applying next-generation sequencing. This genome-wide data also provides a direct measure of replication origin efficiency and can be used to indirectly calculate replication timing.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages239-259
Number of pages21
DOIs
Publication statusPublished - 2018 Jan 1

Publication series

NameMethods in Molecular Biology
Volume1672
ISSN (Print)1064-3745

Keywords

  • Fork restart
  • HTP sequencing
  • Origins
  • Pu-seq
  • rNMP incorporation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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