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DNA‐dependent phosphorylation of histone H2A.X during nucleosome assembly in Xenopus laevis oocytes: involvement of protein phosphorylation in nucleosome spacing.
Author(s) -
Kleinschmidt J.A.,
Steinbeisser H.
Publication year - 1991
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1991.tb07855.x
Subject(s) - phosphorylation , biology , nucleosome , histone , chromatin , xenopus , microbiology and biotechnology , dephosphorylation , protein phosphorylation , histone h1 , histone h2a , dna , biochemistry , phosphatase , protein kinase a , gene
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.