High-Resolution Mapping of Modified DNA Nucleobases Using Excision Repair Enzymes

Methods Mol Biol. 2018;1672:63-76. doi: 10.1007/978-1-4939-7306-4_6.

Abstract

Modification of DNA nucleobases has a profound effect on genome function. We developed a method that maps the positions of the modified DNA nucleobases throughout genomic DNA. This method couples in vitro nucleobase excision with massively parallel DNA sequencing to determine the location of modified DNA nucleobases with single base precision. This protocol was used to map uracil incorporation and UV photodimers in DNA, and a modification of the protocol has been used to map sparse modification events in cells. The Excision-seq protocol is broadly applicable to a variety of base modifications for which an excision enzyme is available.

Keywords: Base excision repair; Circular ligation; High-throughput sequencing; UV photodimers; Uracil incorporation.

MeSH terms

  • DNA / drug effects
  • DNA / genetics*
  • DNA / metabolism*
  • DNA / radiation effects
  • DNA Damage / drug effects
  • DNA Damage / radiation effects
  • DNA Repair*
  • Escherichia coli / genetics
  • High-Throughput Nucleotide Sequencing*
  • Sequence Analysis, DNA
  • Ultraviolet Rays
  • Uracil / pharmacology
  • Yeasts / genetics

Substances

  • Uracil
  • DNA