Structure of polyubiquitinated histone H2A.
We have recently demonstrated that trout liver histones H2A, H2B, and H2A.Z can be polyubiquitinated [Davie, J.R., Delcuve, G.P., Nickel, B.E., Moyer, R., & Bailey, G. (1987) Cancer Res. 47, 5407-5410]. In the present study we determined the arrangement of the ubiquitin molecules in polyubiquitinated histone H2A. Trout liver chromatin fragments. which had histone H1 removed, were digested with Staphylococcus aureus (V8 strain) protease which cleaves specifically on the carboxyl side of glutamic acid residues under the conditions used. The V8 protease readily degraded histone H2A and ubiquitinated (u) H2A at equivalent rates. One site in H2A and uH2A, the peptide bond between Glu 121 and Lys 122, was cleaved, yielding protein species cH2A and cuH2A, respectively. None of the other nucleosomal histones (H2B, H2A.Z, H3, and H4) including uH2B and uH2A.Z were sensitive to digestion. Trout liver histones cleaved with either V8 protease, histone H2A specific protease, or cyanogen bromide were resolved by two-dimensional gel electrophoresis and ubiquitinated peptides detected with anti-ubiquitin IgG. The results suggest that the major arrangement of ubiquitin in polyubiquitinated H2A is a chain of ubiquitin molecules joined to each other by isopeptide bonds to a ubiquitin molecule that is attached to the epsilon-amino group of lysine 119 of histone H2A.
Nickel
B E
BE
Department of Biochemistry, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Davie
J R
JR
D000818:Animals; D002417:Cattle; D002843:Chromatin / Q000032:analysis*; D003488:Cyanogen Bromide; D015180:Electrophoresis, Gel, Two-Dimensional; D006657:Histones / Q000302:isolation & purification*; D008099:Liver / Q000032:analysis*; D010446:Peptide Fragments / Q000302:isolation & purification; D012697:Serine Endopeptidases; D014337:Trout; D014452:Ubiquitins / Q000302:isolation & purification*
D002843:Chromatin; D006657:Histones; D010446:Peptide Fragments; D014452:Ubiquitins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003488:Cyanogen Bromide