DNA-dependent phosphorylation of histone H2A.X during nucleosome assembly in Xenopus laevis oocytes: involvement of protein phosphorylation in nucleosome spacing.

AUTOR(ES)

Kleinschmidt, J A

RESUMO

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.

Documentos Relacionados

• Histone H2A.X gene transcription is regulated differently than transcription of other replication-linked histone genes.
• Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1
• Polyadenylated and 3' processed mRNAs are transcribed from the mouse histone H2A.X gene.
• Dephosphorylation of the C-terminal Tyrosyl Residue of the DNA Damage-related Histone H2A.X Is Mediated by the Protein Phosphatase Eyes Absent*
• Nucleosome assembly in vitro: separate histone transfer and synergistic interaction of native histone complexes purified from nuclei of Xenopus laevis oocytes.