Protein-protein proximity in the association of ribosomal subunits of Escherichia coli: crosslinking of 30 S protein S16 to 50 S proteins by glutaraldehyde or formaldehyde

The interaction of ribosomal subunits from Escherichia coli has been studied using crosslinking reagents. Radioactive ³⁵S-labeled 50 S subunits and non-radioactive 30 S subunits were allowed to reassociate to form 70 S ribosomes. The 70 S particles, containing radioactivity only in the 50 S protein moiety, were incubated with glutaraldehyde or formaldehyde. As a result of this treatment a substantial fraction of the 70 S particles did not dissociate at 1 mm-Mg²⁺. This fraction was isolated and the ribosomal proteins were extracted. The protein mixture was analyzed by the Ouchterlony double diffusion technique by using eighteen antisera prepared against single 30 S ribosomal proteins (all except those against S3, S15 and S17). As a result of the crosslinking procedure it was found that only anti-S16 co-precipitated ³⁵S-labeled 50 S protein. It is concluded that the 30 S protein S16 is at or near the site of interaction between subunits and can become crosslinked to one or more 50 S ribosomal proteins.     

Primary organization of nucleosomes. Interaction of non-histone high mobility group proteins 14 and 17 with nucleosomes, as revealed by DNA-protein crosslinking and immunoaffinity isolation

The binding sites for histones and high mobility group proteins (HMG) 14 and 17 have been located on DNA in the nucleosomal cores and H1/H5-containing nucleosomes. The nucleosomes were specifically associated with two molecules of the non-histone proteins HMG 14 and/or HMG 17 when followed by DNA-protein crosslinking and immunoaffinity isolation of the crosslinked HMG-DNA complexes. HMGs 14 and 17 were shown to be crosslinked in a similar manner to each core DNA strand at four sites: to both 3' and 5' DNA ends and also at distances of about 25 and 125 nucleotides from the 5' termini of the DNA. These sites are designated as HMG(143), (0), (25) and (125). The site HMG(125) is located at the place where no significant histone-DNA crosslinking was observed. The HMG(125) and HMG(25) sites lie opposite one another on the complementary DNA strands across the minor DNA groove and are placed, similarly to histones, on the inner side of the DNA superhelix in the nucleosome. The crosslinking of HMG 17 to the 3' ends of the DNA is much weaker than that of HMG 14. These data indicate that each of two molecules of HMG 14 and/or HMG 17 is bound to the double-stranded core DNA at two discrete sites: to the 3' and 5' ends of the DNA and at a distance of 20 to 25 base-pairs from each DNA terminus inside the nucleosome on a histone-free DNA region. Binding of HMG 14 or 17 does not induce any detectable rearrangement of histones on DNA and both HMGs seem to choose the same sites for attachment in nucleosomal cores and H1/H5-containing nucleosomes.     

乙酰转移酶MYST家族的生物学功能

组蛋白乙酰化是表观遗传修饰的重要方式,主要受到组蛋白乙酰转移酶（histone acetyltransferases, HATs)和组蛋白去乙酰化酶（histone deacetylase, HDACs）催化. MYST是人类HATs的4大家族之一,包括MOF(males absent on the first),TIP60 (tat interacting protein 60 kD),结合ORC1的组蛋白乙酰转移酶（histone acetyltransferase binding to ORC1, HBO1),单核细胞白血病锌指蛋白(monocytic leukemia zinc finger protein, MOZ)和MOZ相关蛋白（MOZ related factor, MORF）等,均具有典型的MYST结构域.MYST介导的乙酰化是重要的翻译后修饰,其催化底物包括组蛋白和非组蛋白,如组蛋白H3, H4, H2A, H2A突变体,以及许多参与DNA代谢、细胞增殖和发育调控的蛋白因子. MYST蛋白家族参与许多细胞的生理过程,本文主要综述其在调节基因转录、DNA损伤修复和肿瘤发生发展等方面的生物学功能.     

Interaction of non-histone proteins HMG1 and HMG2 with core histones in nucleosomes and core particles revealed by chemical cross-linking

Chemical cross-linking was used to study the interaction of the non-histone chromosomal proteins HMG1 and HMG2 with core histones in H1,H5-depleted nucleosomes or core particles. Cross-linking with a 'zero-length' cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and with a longer (cleavable) cross-linker dimethyl-3,3'-dithiobispropionimidate revealed an interaction of HMG1 and HMG2 with (or proximity to) core histones in both types of particles. These results indicated that the presence of the 40-50-base-pairs-long segment of the 'linker' DNA in nucleosomes was not necessary for the establishment of mutual contacts of HMG1 and HMG2 proteins with core histones. Possible implications of the interaction of HMG1 and HMG2 proteins with histones for the structure and functioning of chromatin are discussed.     

Photocrosslinking locates a binding site for the large subunit of human replication protein A to the damaged strand of cisplatin-modified DNA.

The repair proteins XPA, XPC and replication protein A (RPA) have been implicated in the primary recognition of damaged DNA sites during nucleotide excision repair. Detailed structural information on the binding of these proteins to DNA lesions is however lacking. We have studied the binding of human RPA (hRPA) and hRPA-XPA-complexes to model oligonucleo-tides containing a single 1, 3-d(GTG)-cisplatin-modification by photocrosslinking and electrophoretic mobility shift experiments. The 70 kDa subunit of hRPA can be crosslinked with high efficiency to cisplatin-modified DNA probes carrying 5-iodo-2"-deoxyuridin (5-IdU) as crosslinking chromophore. High efficiency crosslinking is dependent on the presence of the DNA lesion and occurs preferentially at its 5"-side. Examination of the crosslinking efficiency in dependence on the position of the 5-IdU chromophore indicates a specific positioning of hRPA with respect to the platination site. When hRPA and XPA are both present mainly hRPA is crosslinked to the DNA. Our mobility shift experiments directly show the formation of a stable ternary complex of hRPA, XPA and the damaged DNA. The affinity of the XPA-hRPA complex to the damaged DNA is increased by more than one order of magnitude as compared to hRPA alone.     

Biochemical and ultrastructural study of the sperm chromatin from Mytilus galloprovincialis

Protein composition and ultrastructure of the mature spermatozoa of the mussel Mytilus galloprovincialis were studied upon gradual decondensation of the nuclei with increasing NaCl concentration. Three types of protein were found, associated with the sperm DNA: (1) the sperm-specific proteins S1, S2 and S3 (80% of the acid-soluble proteins); (2) the four core histones (20%); (3) three non-histone proteins tightly bound to DNA (about 4 micrograms protein per 100 micrograms DNA). The sperm-specific protein S3 was the first to dissociate at about 0.5 M NaCl and electron micrographs of spread nuclei indicated its participation in the final compaction of the nucleus. Hypotonically treated sperm nuclei revealed the presence of 21-25 nm large granules irregularly scattered along some of the DNA fibers. These granules correspond to the 'superbeads' of histone-containing chromatins. The tightly bound non-histone proteins were represented by a triplet in the range 60-80 kD. They formed 30-60 nm large annular bodies holding DNA fibers and resisting high salt-detergent treatment.     

The synthesis and decay of histone fractions and of deoxyribonucleic acid in the developing avian brain

1. The turnover of cerebral histones and DNA after injection of [4,5-(3)H]leucine or [methyl-3-(3)H]thymidine, respectively, was studied in the developing chick. 2. Chromatin was prepared from chick nuclei that had been purified by centrifugation through 1.9m-sucrose. 3. Nuclear proteins were fractionated into three major histone classes, F1 (lysine-rich), F2(b) (slightly lysine-rich) and [F3+F2(a)] (arginine-rich), and a non-histone protein residue. 4. The proportions of the histone classes remained constant throughout the period of development studied. 5. All histone fractions decayed at a similar rate, initially with a half-life of around 5 days, later with a half-life of 19 days. 6. Non-histone proteins from chromatin decayed in a heterogeneous manner with a wide range of half-lives. 7. Short-term labelling studies showed that all histone fractions were synthesized at the same rate. 8. Some non-histone proteins were very rapidly synthesized relative to histones. 9. DNA had a longer half-life than any histone fraction studied. A biphasic exponential decay curve with half-lives of 23 and 50 days was found. 10. It was concluded that the turnover of histones can occur independently of that of DNA and that different histone classes have similar rates of synthesis and decay.     

Conformational studies of two non-histone chromosomal proteins and their interactions with DNA.

The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied. 1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding. 3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA.     

A new cAMP independent protein kinase tightly bound to DNA, in rat liver nuclei

A protein kinase has been characterized among the proteins tightly bound to DNA. It is not extracted with 1 M NaCl and is released by extensive DNase I digestion. This enzyme is able to phosphorylate nucleosomal histones, essentially H2B and H3, and several non-histone proteins associated with DNA, on serine residue(s). It does not phosphorylate protamine, casein, phosvitin and the chromosomal non-histone proteins extracted with 1 M NaCl and is cAMP independent. This protein kinase can be distinguished from the previously described enzymes.     

The enhancers and promoters of the Xenopus laevis ribosomal spacer are associated with histones upon active transcription of the ribosomal genes

The presence of histones on the enhancer-promoter region of the X.laevis ribosomal spacer has been studied in embryos at stage 40, where the ribosomal genes are actively transcribed. Isolated tadpole nuclei were either fixed with formaldehyde or irradiated with UV laser to crosslink histones to DNA. The purified protein-DNA complexes were immunoprecipitated with antibodies to the histones H1, H2A and H4 and the DNA fragments carrying the respective histones were analyzed for the presence of spacer enhancer-promoter sequences by hybridization to specific DNA probe. The two independent crosslinking procedures revealed the presence of these DNA sequences in the precipitated DNA. The quantitative analysis of the UV laser-crosslinked complexes showed that histones H2A and H4 were associated with enhancer-promoter DNA in amounts similar to those found for bulk DNA, whilst the content of H1 was reduced.     

Interaction of non-histone proteins with DNA and chromatin from Drosophila and mouse cells. Specificity, isolation and analysis of complexes.

The specificity of the binding of purified non-histone proteins to DNA has been investigated through two types of experiments. Using a nitrocellulose filter assay at a low protein/DNA ratio, the binding of mouse non-histone proteins to mouse DNA was twice as great as the binding of mouse non histone protein to Drosophila DNA. The reverse experiment using Drosophila non-histone protein confirmed the interpretation that some protein . DNA complexes were specific. Protein . DNA complexes isolated by gel filtration chromatography indicated that 20% or 10% of the non-histone protein was bound to homologous or heterologous DNA respectively. Purified non-histone proteins bound with lower efficiency (15%) than unpurified but with higher specificity to soluble chromatin than to naked DNA. This binding did not result from an exchange between chromatin non-histone proteins and purified non-histone proteins added in excess. DNA-bound and chromatin-bound proteins were analysed on polyacrylamide gels. Whereas no major qualitative differences were observed with DNA-bound proteins, some proteins bound to homologous mouse chromatin were different from those bound to heterologous Drosophila chromatin. These results suggest a possible role of DNA-bound non-histone proteins in the regulation of gene expression.     

La surexpression de l'androgen-binding protein (ABP) provoque des modifications morphologiques et fonctionnelles dans le testicule de souris

The Sertoti cells (SC) of many species produce an androgen-binding protein (ABP) which is secreted into both the blood and lumen of the seminiferous tubule. In the latter, it is transported to the epididymis where is taken up by epithelial cells, and is thought to play a role in sperm maturation. In view of the importance of ABP, we thought it would be pertinent to make several transgenic mice (TM) lines bearing the rABP gene to unravel its role in male reproductive physiology. A 5.5 Kb rat genomic DNA clone was microinjected into the pronucleus of fertilized mouse ova which were subsequently implanted into the oviduct of pseudopregnant CD-1 female mice. Detection of TM was performed by Southern Blot and PCR analysis using respectively, a 32p labeled rABP cDNA probe and oligonucleotides recognizing exons 1 and 7 of rABP gene. Chromosomic localization of the transgene was carried out by fluorescent in situ hybridization (FISH) in metaphasic cells obtained from bone marrow of TM. rABP expression was analyzed by Northern blot and RT-PCR techniques in most tissues of heterozigote TM. In the testis, specific cell expression was determined by in situ hybridization (ISH) and protein localization by immunohistochemistry. ABP-binding activity was performed by the carbon dextran method and analysis of protein internalization by autohistoradiographic detection of a (3H) testosterone-ABP complex. DNA fragmentation was investigated by the TUNEL technique and by electrophoresis of total genomic DNA. Testicular and epididymal morphology was studied by light and electron microscopy. Two offspring carrying the rABP gene were identified by Southern blotting, and two lines of mice (designated ABP7 and ABP24) were generated by selective breeding of the male founders with normal B6D2F1 females. FISH analysis demonstrated a different chromosomal localization of the transgene in both lines. Both rABP transgenic pedigrees presented reduced fertility. Northern blot and RT-PCR studies showed overexpression of rABP mRNA in the testis. ovary and uterus in ABP24 and ABP7 transgenic lines. rABP mRNA was appropriately expressed in SC as demonstrated by ISH. DHT-sH binding of testicular homogenates was increased 10 fold in TM compared to controls. In adult testis of TM, some seminiferous tubules showed disorganization of the epithelium, increased number of SC, presence of vacuoles, germ cell meiotic arrest and germ cell degeneration. DNA fragmentation was demonstrated in germ cells during meiosis. rABP protein was localized in the intersticial space, and into some tubules, in SC and germ cells at different steps of maturation. rABP internalization was strongly increased in both germ and epididymal cells in TM. The present results reinforce the increasing evidence of the role of ABP during spermatogenesis, even though further experiments are required to unravel its definitive implication in testicular and epidididymal homeostasis.     

Interaction of the non-histone protein PS1 with some chromatin components

The binding of non-histone protein from mouse spleen chromatin located in the sites highly sensitive to micrococcal nuclease and DNA-ase I, to DNA and histones was studied. The binding of the DNA-protein complexes to nitrocellulose filters demonstrated the absence of protein binding to DNA. A highly selective binding of protein PS1 to histones H1 and H2A and to one of the non-histone proteins (presumably HMG 14) was revealed. It is concluded that protein PS1 is incorporated into chromatin by the protein-protein interactions.     

Porcine follitropin. The amino-acid sequence of the beta subunit

By treatment with tRNA in the presence of 1 mM MgCl2, a chromatin preparation was obtained containing all five major histone fractions but lacking a considerable portion of non-histone proteins. This chromatin preparation as well as chromatin extracted with 0.6 M NaCl (depleted of H1 histone and some non-histone proteins) were characterized in respect of solubility and chromatin DNA accessibility. Both samples possessed practically the same solubility in the presence of 0.15 M NaCl and 1 mM MgCl2. The solubility of tRNA-treated chromatin in 5 and 10 mM MgCl2 was higher than that of salt-extracted chromation. The accessibility of the DNA of these chromatin preparations was tested with DNA-dependent RNA polymerase of Escherichia coli as a probe, using procedure that permits measurement of binding site frequency. Both tRNA-treated and salt-extracted chromatin contained as many as 33% and untreated chromatin as few as 4% of the number of binding sites found on protein-free DNA. These results demonstrate that at least in part the non-histone proteins are responsible for salt-induced insolubility and low DNA accessibility of chromatin, thus revealing the importance of non-histone proteins in the maintenance of an overall chromatin structure.     

Protein kinases and their protein substrates associated with chromatin and ribosomes in SV40-transformed rat cells.

The level of endogenous protein phosphorylation in non-histone chromosomal and ribosomal wash proteins is 7--10 times greater in SV40-transformed rat cells than in untransformed parental cells. Protein kinase activity in these proteins was fractionated by either phosphocellulose or DEAE-cellulose chromatography. One major and one minor component were detected in non-histone proteins and only one component in ribosomal wash proteins when the activity in each fraction was measured with an exogenous substrate, casein. These enzymes prefer casein to whole histone as substrate and are cyclic AMP-independent. The enzyme activity in a major peak of non-histone proteins and in ribosomal wash proteins measured with casein as substrate is 3 times greater in transformed cells than in untransformed cells, whereas pH optimum, cation requirements and apparent Km values for casein and ATP are identical or very similar in the two cell types. No significant phosphatase was detected in non-histone and ribosomal wash proteins from the two types of cell. The patterns of endogenous protein phosphorylation in these protein fractions analysed by gel electrophoresis are significantly different between these cells. These results suggest that the high level of endogenous protein phosphorylation in non-histone and ribosomal wash proteins from SV40-transformed cells is caused mainly by the increased activity of protein kinase and the nature of protein substrates.     

The structure of the complexes of DNA with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions. I. Circular dicroism spectroscopy

The mechanisms of interaction of the non-histone chromosomal protein HMGB1 and linker histone H1 with DNA have been studied using circular dichroism and absorption spectroscopy. Both of the proteins are located in the inter-nucleosomal regions of chromatin. It was demonstrated that properties of the DNA-protein complexes depend on the protein content and can not be considered as a simple summing up of the effects of individual protein components. Interaction of HMGB1 and H1 proteins is shown to be co-operative rather than competitive. Lysine-rich histone H1 facilitates the binding of the HMGB1 with DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino-acid residues in the C-terminal domain of the HMGB1 protein. The observed joint action of the and H1 proteins stimulates DNA condensation with formation of the anisotropic DNA-protein complexes with typical psi-type CD spectra. Structural organization of the complexes depends not only on the DNA-protein interactions, but also on the interaction between HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the character of interactions between the components in the triple DNA-HMGB1-H1 complex. Binding of Mn2+ ions causes the weakening of the DNA-protein interactions and strengthening the protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.