Actinomycin D induces histone gamma-H2AX foci and complex formation of gamma-H2AX with Ku70 and nuclear DNA helicase II.
Formation of gamma-H2AX foci is a P. O.cellular response to genotoxic stress, such as DNA double strand breaks or stalled replication forks. Here we show that gamma-H2AX foci were also formed when cells were incubated with 0.5 microg/ml DNA intercalating agent actinomycin D. In untreated cells, gamma-H2AX co-immunoprecipitated with Ku70, a subunit of DNA-dependent protein kinase, as well as with nuclear DNA helicase II (NDH II), a DEXH family helicase also known as RNA helicase A or DHX9. This association was increased manifold after actinomycin D treatment. DNA degradation diminished the amount of Ku70 associated with gamma-H2AX but not that of NDH II. In vitro binding studies with recombinant NDH II and H2AX phosphorylated by DNA-dependent protein kinase confirmed a direct physical interaction between NDH II and gamma-H2AX. Thereby, the NDH II DEXH domain alone, i.e. its catalytic core, was able to support binding to gamma-H2AX. Congruently, after actinomycin D treatment, NDH II accumulated in RNA-containing nuclear bodies that predominantly co-localized with gamma-H2AX foci. Taken together, these results suggest that histone gamma-H2AX promotes binding of NDH II to transcriptionally stalled sites on chromosomal DNA.
Mischo
Hannah Elisabeth
HE
Departments of Biochemistry and Molecular Biology, Institute of Molecular Biotechnology, Postfach 100 813, D-07708 Jena, Germany.
Hemmerich
Peter
P
Grosse
Frank
F
Zhang
Suisheng
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D034961:Antigens, Nuclear / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology*; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D010750:Phosphoproteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D010750:Phosphoproteins; D011994:Recombinant Proteins; D003609:Dactinomycin; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen