DNA Fiber Analysis: Mind the Gap!

Annabel Quinet, Denisse Carvajal-Maldonado, Delphine Lemacon, Alessandro Vindigni

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

65 Scopus citations

Abstract

Understanding the mechanisms of replication stress response following genotoxic stress induction is rapidly emerging as a central theme in cell survival and human disease. The DNA fiber assay is one of the most powerful tools to study alterations in replication fork dynamics genome-wide at single-molecule resolution. This approach relies on the ability of many organisms to incorporate thymidine analogs into replicating DNA and is widely used to study how genotoxic agents perturb DNA replication. Here, we review different approaches available to prepare DNA fibers and discuss important limitations of each approach. We also review how DNA fiber analysis can be used to shed light upon several replication parameters including fork progression, restart, termination, and new origin firing. Next, we discuss a modified DNA fiber protocol to monitor the presence of single-stranded DNA (ssDNA) gaps on ongoing replication forks. ssDNA gaps are very common intermediates of several replication stress response mechanisms, but they cannot be detected by standard DNA fiber approaches due to the resolution limits of this technique. We discuss a novel strategy that relies on the use of an ssDNA-specific endonuclease to nick the ssDNA gaps and generate shorter DNA fibers that can be used as readout for the presence of ssDNA gaps. Finally, we describe a follow-up DNA fiber approach that can be used to study how ssDNA gaps are repaired postreplicatively.

Original languageEnglish
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages55-82
Number of pages28
DOIs
StatePublished - 2017

Publication series

NameMethods in Enzymology
Volume591
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • DNA fiber analysis
  • DNA replication
  • Postreplication repair
  • Replication stress
  • S1 nuclease
  • Single-stranded DNA gap

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