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Tryptic digestion of chicken erythrocyte nuclei, to a level at which no intact histone remained, resulted in a set of resistant peptides. These were partially separated by exclusion chromatography. One of the peptides was shown to represent the central sequence 12-118 of histone H2A. This was established by amino acid analysis and by Edman degradations. Comparison of the sequence of histone H2A from a wide range of cell types shows that the tryptic cleavage points correspond closely to the limits of the highly conserved central sequence and not to the limits of the strongly basic regions. It is proposed that the 11 N-terminal and 10 C-terminal residues cleaved by trypsin are exposed in chromatin and play a structural and functional role different from the central 107 residues. The exposed position of the 118-119 bond accords with the known linkage point of ubiquitin to residue 119 of histone H2A in the semi-histone A24.Proteases are valuable structural probes in the study of chromatin and test for the relative acessibility of histone amino acid residues in the structure. Trypsin has been widely used [l -51 and it has been shown that histones H1 and H5 are the first to be digested, whilst the core histones are digested in the order H3, H2A, H4, H2B to give a distinct and fairly reproducible limit digest pattern. On the basis of tyrosine labelling [l] and amino acid compositions [4] of the unseparated limit digest mixture, it was postulated that it represents a mixture of C-terminal peptides that have lost between 23 and 42 N-terminal residues [4]. Limit fragments of histones H3 and H4 generated by an endogenous trypsin-like activity in cycad pollen nuclei have been identified by sequencing as H3(24-135) and H4(17-102) [6]. Neither histone had lost C-terminal residues. Although the enzyme responsible is not known, this result suggests that significantly less than 30 residues may be involved in the N-terminal domains.Trypsin digestions of chromatin and core particles carried out at the high salt concentrations in which the protein is dissociated from the DNA give rise to similar limiting products, indicating that the protection conferred on the C-terminal portions is due primarily to the conformation of the histones in the core complex and not directly attributable to the DNA [2].The high capability of the histone limit fragments to act as core particle DNA organisers has been illustrated by the finding that core particles containing no intact histones have a sedimentation coefficient of 9.7 S [5] or 9.0 S [4] as compared to 11.3 S for the complete core particle. If the histone mass loss were 20% this would cause a reduction to about 10.5 S and if some N-terminal residues of H2A and H2B are disordered in the core particle, as suggested from NMR experiments [7], then a 9.7-S particle might be little changed in overall conformation. A very recent publication indicates that trypsin digestion reduces the sedimentation of a core particle only from 10.4 S to 10.2 S [26]. Whitlock and Simpson [4] have also shown that th...

Biochemical studies have implicated both nucleosome core assembly and/or the subsequent binding of the linker histone H1 in transcriptional repression (4 -9). Because the affinities of sequence-specific DNA-binding proteins for nucleosomal DNA are often dramatically reduced compared with free DNA (reviewed in Ref. 10), nucleosomes repress the transcriptional process, at least in part, by inhibiting access of activator proteins to their cognate binding sites in chromatin. Factor occupancy of binding sites in nucleosomal arrays is most likely achieved through multiple mechanisms involving nucleosome disruption, nucleosome displacement in trans, or histone octamer sliding in cis (reviewed in Ref. 11). The idea of mobile nucleosomes (i.e. nucleosome sliding) is attractive in that it has the potential to create transient accessibility of factors to their binding sites. Its potential importance is further implicated in studies that illustrate the differential affinity of factors for sites at different translational and rotational positions within nucleosomes (12-15). Using a model system consisting of chromatin assembled on tandemly repeated sea urchin 5 S rRNA nucleosome positioning sequences (16), Bradbury and colleagues (17, 18) have previously reported that nucleosomes adopt a dominant position surrounded by minor positions 10 base pairs apart (i.e. in the same rotational frame). These data indicate that the cluster of octamer positions is in dynamic equilibrium in low ionic strength conditions. In the presence of the linker histone H1, this mobility is inhibited (19). The same group found that this short range sliding behavior also applies to bulk mononucleosomes and nucleosomes reconstituted onto sequences of the Alu family of ubiquitous repeats. Thus, they proposed that nucleosome mobility is a general behavior (20). Moreover, H1-mediated reduction in nucleosome mobility has been implicated in repression of transcription of a dinucleosome reconstituted onto a dimerized Xenopus somatic 5 S rRNA gene (2).In our previous studies, we reported for the first time direct inhibition of factor binding by the association of H1 with nucleosome cores. The binding of H1 to form a chromatosome significantly repressed the subsequent binding of USF 1 but only slightly inhibited GAL4-AH binding (1). In this report, we extend these studies to explore the underlying mechanisms by which H1 repressed USF binding. The results illustrate H1-mediated repression of USF binding to a stably positioned nucleosome. Thus, H1 repression in this instance occurred in the absence of nucleosome mobility. In addition, H1 repressed USF binding to a site on the opposite side of the histone octamer from the entry and exit points of the linker DNA. These data are consistent with a mechanism by which H1 stabilizes histone octamer-DNA interactions, thus reducing transient exposure of factor binding sites. MATERIALS AND METHODSPreparation of DNA Probes-The 183-bp probe DNAs, referred to as GU or UG probes, were either directly generated by BamHI digesti...

It is generally assumed that radiation-induced micronuclei (MN) in cytokinesis-blocked cells are an expression of cellular radiosensitivity. Therefore, radiosensitive cells should have a high frequency of MN and radioresistant cells should show lower levels. We have irradiated cells of a panel of 13 neuronal cell lines of widely differing radiosensitivity [human neuroblastomas: N2alpha, SHSY5Y, SK-N-SH, KELLY and SK-N-BE(2c); murine neuroblastomas: OP-6 and OP-27; human glioblastomas: G120, G60, G28, G112, G44 and G62] and compared their radiation response using the micronucleus and standard clonogenic assays. It was found that micronucleus frequency was much higher in some of the radioresistant cell lines (N2alpha, G28, G120 and G44; SF2 >/= 0.60). These cell lines showed a high frequency of more than 0.32 MN per gray of (60)Co gamma radiation per binucleated cell. On the other hand, the more radiosensitive cell lines (OP-27 and SK-N-SH, SF2