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Genetic and Molecular Approaches to Study Chromosomal Breakage at Secondary Structure–Forming Repeats

  • Anissia Ait Saada
  • Alex B. Costa
  • Kirill S. LobachevEmail author
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Part of the Methods in Molecular Biology book series (MIMB, volume 2153)

Abstract

DNA repeats capable of adopting stable secondary structures are hotspots for double-strand break (DSB) formation and, hence, for homologous recombination and gross chromosomal rearrangements (GCR) in many prokaryotic and eukaryotic organisms, including humans. Here, we provide protocols for studying chromosomal instability triggered by hairpin- and cruciform-forming palindromic sequences in the budding yeast, Saccharomyces cerevisiae. First, we describe two sensitive genetic assays aimed to determine the recombinogenic potential of inverted repeats and their ability to induce GCRs. Then, we detail an approach to monitor chromosomal DSBs by Southern blot hybridization. Finally, we describe how to define the molecular structure of DSBs. We provide, as an example, the analysis of chromosomal fragility at a reporter system containing unstable Alu-inverted repeats. By using these approaches, any DNA sequence motif can be assessed for its breakage potential and ability to drive genome instability.

Key words

Inverted repeats Secondary structures Genome instability DSB detection Gross chromosomal rearrangements 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Anissia Ait Saada
    • 1
  • Alex B. Costa
    • 1
  • Kirill S. Lobachev
    • 1
    Email author
  1. 1.School of Biological Sciences, Institute for Bioengineering and BioscienceGeorgia Institute of TechnologyAtlantaUSA

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