Studying Single-Stranded DNA Gaps at Replication Intermediates by Electron Microscopy

Jessica Jackson, Alessandro Vindigni

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

2 Scopus citations

Abstract

Single-stranded DNA gaps are frequent structures that accumulate on newly synthesized DNA under conditions of replication stress. The identification of these single-stranded DNA gaps has been instrumental to uncover the mechanisms that allow the DNA replication machinery to skip intrinsic replication obstacles or DNA lesions. DNA fiber assays provide an essential tool for detecting perturbations in DNA replication fork dynamics genome-wide at single molecule resolution along with identifying the presence of single-stranded gaps when used in combination with S1 nuclease. However, electron microscopy is the only technique allowing the actual visualization and localization of single-stranded DNA gaps on replication forks. This chapter provides a detailed method for visualizing single-stranded DNA gaps at the replication fork by electron microscopy including psoralen cross-linking of cultured mammalian cells, extraction of genomic DNA, and finally enrichment of replication intermediates followed by spreading and platinum rotary shadowing of the DNA onto grids. Discussion on identification and analysis of these gaps as well as on the advantages and disadvantages of electron microscopy relative to the DNA fiber technique is also included.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages81-103
Number of pages23
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2444
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • DNA replication
  • DNA replication stress
  • Electron microscopy
  • Replication structures
  • ssDNA gaps

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