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Physical and Genetic Assays for the Study of DNA Joint Molecules Metabolism and Multi-invasion-Induced Rearrangements in S. cerevisiae.

Authors
  • Piazza, Aurèle1, 2, 3
  • Rajput, Pallavi2
  • Heyer, Wolf-Dietrich4, 5
  • 1 Spatial Regulation of Genomes, Institut Pasteur, UMR3525 CNRS, Paris, France. , (France)
  • 2 Department of Microbiology and Molecular Genetics, University of California, Davis, CA, USA.
  • 3 Univ Lyon, ENS, UCBL, CNRS, INSERM, Laboratory of Biology and Modelling of the Cell, UMR5239, Lyon, France. , (France)
  • 4 Department of Microbiology and Molecular Genetics, University of California, Davis, CA, USA. [email protected]
  • 5 Department of Molecular and Cellular Biology, University of California, Davis, CA, USA. [email protected]
Type
Published Article
Journal
Methods in molecular biology
Publisher
Clifton, N.J. : Humana Press
Publication Date
Jan 01, 2021
Volume
2153
Pages
535–554
Identifiers
DOI: 10.1007/978-1-0716-0644-5_36
PMID: 32840803
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

DNA double-strand breaks (DSBs) are genotoxic lesions that can be repaired in a templated fashion by homologous recombination (HR). HR is a complex pathway that involves the formation of DNA joint molecules (JMs) containing heteroduplex DNA. Various types of JMs are formed throughout the pathway, including displacement loops (D-loops), multi-invasions (MI), and double Holliday junction intermediates. Dysregulation of JM metabolism in various mutant contexts revealed the propensity of HR to generate repeat-mediated chromosomal rearrangements. Specifically, we recently identified MI-induced rearrangements (MIR), a tripartite recombination mechanism initiated by one end of a DSB that exploits repeated regions to generate rearrangements between intact chromosomal regions. MIR occurs upon MI-JM processing by endonucleases and is suppressed by JM disruption activities. Here, we detail two assays: a physical assay for JM detection in Saccharomyces cerevisiae cells and genetic assays to determine the frequency of MIR in various chromosomal contexts. These assays enable studying the regulation of the HR pathway and the consequences of their defects for genomic instability by MIR.

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