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Analysis of Double-Strand Break Repair by Nonhomologous DNA End Joining in Cell-Free Extracts from Mammalian Cells

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Molecular Toxicology Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1105))

Abstract

Double-strand breaks (DSB) in genomic DNA are induced by ionizing radiation or radiomimetic drugs but also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair which is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient cell lines yielded insight into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay which permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.

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Abbreviations

ATP:

Adenosine triphosphate

BPB:

Bromophenol blue

BSA:

Bovine serum albumin

conc.:

Concentration

dNTP:

Deoxy-nucleoside triphosphate

DSB:

Double-strand break

DTT:

Dithiothreitol

EDTA:

Ethylenediamine-tetra-acetate

EGTA:

Ethyleneglycol-bis (β-amino-ethyl) ether-tetra-acetate

EthBr:

Ethidium bromide

EtOH:

Ethanol

HRR:

Homologous recombination repair

IR:

Ionizing radiation

MOPSO:

3-(N-Morpholino)-2-hydroxy-propanesulfonic acid

NHEJ:

Nonhomologous DNA end joining

O/N:

Overnight

PCV:

Packed cell volume

PMSF:

Phenyl-methyl-sulfonyl-fluoride

ddH2O:

Double-distilled water

RE:

Restriction endonuclease

RT:

Room temperature

SDS:

Sodium dodecyl sulfate

SSA:

Single-strand annealing

UV:

Ultra violet

XC:

Xylene cyanol

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Author information

Authors and Affiliations

  1. Department of Pediatric Oncology, University Children’s Hospital Essen, Hufelandstr. 55, 45122, Essen, Germany

    Steffi Kuhfittig-Kulle

  2. Institute of Genetics, University of Essen, Essen, Germany

    Petra Pfeiffer, Andrea Odersky & Wolfgang Goedecke

Authors
  1. Petra Pfeiffer
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  2. Andrea Odersky
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  3. Wolfgang Goedecke
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  4. Steffi Kuhfittig-Kulle
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Corresponding author

Correspondence to Steffi Kuhfittig-Kulle .

Editor information

Editors and Affiliations

  1. Occupational Health, University of Pittsburgh Dept. Environmental &, Pittsburgh, Pennsylvania, USA

    Phouthone Keohavong

  2. Dept. of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, Fort Lauderdale, Florida, USA

    Stephen G. Grant

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© 2014 Humana Press

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Pfeiffer, P., Odersky, A., Goedecke, W., Kuhfittig-Kulle, S. (2014). Analysis of Double-Strand Break Repair by Nonhomologous DNA End Joining in Cell-Free Extracts from Mammalian Cells. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_39

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  • DOI: https://doi.org/10.1007/978-1-62703-739-6_39

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-738-9

  • Online ISBN: 978-1-62703-739-6

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