Characterization of poly(ADP-ribose)--histone H1 complex formation in purified polynucleosomes and chromatin.
Poly(ADP-ribose) [poly(ADP-Rib)] polymerase of HeLa nucleosomes has been shown in vitro, to catalyze the synthesis of a complex of histone H1 containing 2 H1 histones and 15-16 units of oligo(ADP-Rib). The synthesis of the H1 complex in vitro was compared in polynucleosome populations of various sizes (3--16 and greater than 30) released from HeLa nuclei following micrococcal nuclease digestion. Poly(ADP-Rib) was synthesized from [32P]NAD and the poly(ADP-ribosyl)ation of H1 was studied by selective H1 extraction, gel electrophoresis and autoradiography. Quantitative differences in H1 complex formation occurred when either chromatin concentration or polynucleosome length was varied. The data indicated that H1 complex formation in vitro was favored in polynucleosomes 16 nucleosomes long as compared to 8 nucleosomes. A series of partially ADP-ribosylated H1 species was also detected. Partially modified H1 species migrate more slowly than pure H1 in dodecylsulfate gels. The reduced mobility is a function of the number of attached ADP-Rib moieties. Thus, molecules containing one molecule of H1 and various numbers of ADP-Rib residues can be separated. When the partially modified H1 species were incubated in alkali to cleave the linkage of ADP-Rib to protein, (ADP-Rib1-15) were detected by chain length analysis on 15% polyacrylamide gels. The intermediate H1 species could be chased, in vitro, into as H1 complex with NAD and thus were determined to be successive precursors in the formation of the H1 complex. Evidence is presented that the H1 complex is synthesized in intact cells permeabilized with lysolecithin.
D002843:Chromatin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D046911:Macromolecular Substances; D008836:Micrococcal Nuclease / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis / Q000378:metabolism*
D002843:Chromatin; D006657:Histones; D046911:Macromolecular Substances; D009702:Nucleoside Diphosphate Sugars; D009707:Nucleosomes; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D008836:Micrococcal Nuclease