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Analysis of S100A11 in DNA Damage Repair

  • Franziska Foertsch
  • Christian MelleEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

Abstract

DNA damage possesses the capacity to threaten the genomic integrity of an organism. A multitude of proteins are involved in the detection and repair of DNA double-strand breaks (DSBs), a severe kind of DNA damage. The function of DNA repair proteins can be examined by biochemical assays in vitro as well as in cell-based studies. The Ca2+-binding protein S100A11 shows functional interactions with factors involved in the repair of DSBs by homologous recombination (HR), a high-fidelity DNA repair pathway, such as RAD51 and RAD54B. The key enzyme of the homologous recombination repair is RAD51 that catalyzes the invasion of single-stranded DNA (ssDNA) into double-stranded DNA (dsDNA) containing homologous regions and the exchange of these DNA molecules generating heteroduplex DNA (hDNA). In this chapter, we describe a protocol for the purification of S100A11 to near homogeneity. Using purified proteins, we show the ability of S100A11 to stimulate RAD51 in a DNA strand exchange assay. Additionally, we describe a protocol how S100A11 can be localized in sites of DNA repair by immunofluorescence staining. Furthermore, we present a protocol for assessment of chromosomal aberrations after depletion of S100A11 that illustrate the apparent involvement of S100A11 in genome integrity.

Key words

DNA damage Double-strand breaks Chromosomal aberrations Repair foci S100A11 

Notes

Acknowledgments

We are grateful to Drs Anna Szambowska (FLI Jena) for purified RPA and Anja Weise (UKJ) for microscopic assessment of chromosomal aberrations.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Biomolecular Photonics GroupJena University HospitalJenaGermany

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