DNA-dependent phosphorylation of histone H2A.X during nucleosome assembly in Xenopus laevis oocytes: involvement of protein phosphorylation in nucleosome spacing.
ATP is required for physiological nucleosome alignment in chromatin reconstituted from high-speed nuclear supernatants of Xenopus laevis oocytes. Here we show that during in vitro nucleosome assembly the histone variant H2A.X becomes phosphorylated upon transfer onto DNA, a process which is also observed in vivo. Histone H2A.X phosphorylation increases in the early phase of the assembly reaction, reaching a steady state after approximately 16 min and is maintained with a half-life of the phosphate groups of approximately 2 h. After 6 h, the overall phosphorylation state of H2A.X is reduced, indicating that the phosphorylation-dephosphorylation ratio decreases considerably over time. Addition of alkaline phosphatase leads to a persistently lowered state of H2A.X phosphorylation, in contrast to other nuclear phosphoproteins which undergo rapid rephosphorylation. This suggests that H2A.X phosphorylation is a unique step in the histone-to-DNA transfer process. Selective inhibition of DNA-dependent phosphorylation of H2A.X and of other proteins causes a loss of the physiological 180 bp spacing.
Institut für Virusforschung Deutsches Krebsforschungszentrum, Heidelberg, FRG.
J A
JA
Kleinschmidt
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H
Steinbeisser
D000818:Animals; D002843:Chromatin; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000378:metabolism*; D008845:Microinjections; D009707:Nucleosomes; D009865:Oocytes / Q000166:cytology; D010766:Phosphorylation; D010957:Plasmids; D014982:Xenopus laevis
D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D004247:DNA