José M. Sogo

Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle

The structure of ribosomal chromatin in exponentially growing Friend cells, in stationary cells, and in metaphase chromosomes was studied by psoralen photocrosslinking. It is shown that in intact cells, two distinct types of ribosomal chromatin coexist in Friend cells, one that contains nucleosomes and represents the inactive copies and one that lacks a repeating structure and corresponds to the transcribed genes. A single gene copy is either in one or the other chromatin state. The relative amounts of the two types of structures are similar in interphase and metaphase, however, their run-on activities differ significantly. This suggests that the two states of chromatin are maintained independently of the transcriptional process and that they are stably propagated through the cell cycle.

Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures.

The structure of replicating simian virus 40 (SV40) minichromosomes was studied by DNA crosslinking with trimethyl-psoralen. The procedure was used both in vitro with extracted SV40 minichromosomes as well as in vivo with SV40-infected cells. Both procedures gave essentially the same results. Mature SV40 minichromosomes are estimated to contain about 27 nucleosomes (error +/- 2), except for those molecules with a nucleosome-free gap, which are interpreted to contain 25 nucleosomes (error +/- 2). In replicative intermediates, nucleosomes are present in the unreplicated parental stem with the replication fork possibly penetrating into the nucleosomal DNA before the histone octamer is removed. Nucleosomes reassociate on the newly replicated DNA branches at distances from the branch point of 225 ( +/- 145) nucleotides on the leading strand and of 285( +/- 120) nucleotides on the lagging strand. In the presence of cycloheximide, daughter duplexes contained unequal numbers of nucleosomes, supporting dispersive and random segregation of parental nucleosomes. These were arranged in clusters with normal nucleosome spacing. We detected a novel type of interlocked dimer comprising two fully replicated molecules connected by a single-stranded DNA bridge. We cannot decide whether these dimers represent hemicatenanes or whether the two circles are joined by a Holliday-type structure. The joining site maps within the replication terminus. We propose that these dimers represent molecules engaged in strand segregation.

Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks.

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.

Histone acetylation facilitates RNA polymerase II transcription of the Drosophila hsp26 gene in chromatin.

A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for those acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell‐free system to reconstitute chromatin with increased histone acetylation levels, we directly tested for a causal role of histone acetylation in transcription by RNA pol II. Chromatin, containing either control or acetylated histones, was reconstituted to comparable nucleosome densities and characterized by electron microscopy after psoralen cross‐linking as well as by in vitro transcription. While H1‐containing control chromatin severely repressed transcription of our model hsp26 gene, highly acetylated chromatin was significantly less repressive. Acetylation of histones, and particularly of histone H4, affected transcription at the level of initiation. Monitoring the ability of the transcription machinery to associate with the promoter in chromatin, we found that heat shock factor, a crucial regulator of heat shock gene transcription, profited most from histone acetylation. These experiments demonstrate that histone acetylation can modulate activator access to their target sites in chromatin, and provide a causal link between histone acetylation and enhanced transcription initiation of RNA pol II in chromatin.

Characterization of a protein covalently linked to the 5′ termini of the DNA of Bacillus subtilis phage φ29

Abstract We have isolated a covalent DNA-protein complex from bacteriophage φ29 particles. Polyacrylamide gel electrophoresis and tryptic peptide analysis showed that the protein present in the complex is very similar or identical to p3, an early induced protein essential for viral DNA replication. When the DNA-protein complex is treated with the restriction endonuclease Eco RI, the protein is specifically associated to the two terminal fragments, A and C. The protein is probably linked to the 5′ termini of the DNA since proteinase K-treated DNA is resistant to phosphorylation with polynucleotide kinase, even after treatment with alkaline phosphatase, while it is sensitive to exonuclease III. By electron microscopy the protein is visualized as a dot located at the ends of unit length DNA molecules. Mixed infection of Bacillus subtilis , at 42 °C, with ts † mutants in cistrons 2 and 3 only produces ts 2 progeny. This finding suggests that an inactive protein p3 bound to the DNA of the ts 3 mutant is not replaced by a functional protein and, as a consequence, replication of the ts 3 DNA does not occur.

RNA Polymerase binding sites and transcription map of the DNA of Bacillus subtilis phage φ29

Abstract The Bacillus subtilis RNA polymerase bound to phage φ29 DNA has been visualized by electron microscopy. Seven specific binding sites have been observed at 1.7(±0.4) † , 25.5(±0.5), 26.7(±0.4), 59.4(±1.2), 79.3(±0.9), 91.3(±0.6) and 99sd3(± 0.4) DNA length units (one unit is equal to 1% of the length of φ29 DNA). Three of the sites are located in fragment Eco RI A, two in fragment Eco RI B and two in fragment Eco RI C. The same binding sites are seen whether proteinase K-treated φ29 DNA or protein p3-containing φ29 DNA is used. By hybridization of early or late φ29 induced RNA, at saturation, to the separated strands of uniformly labelled φ29[ 32 P]DNA restriction fragments, which cover more than 95% of the genome, we have determined the extent of the in vivo early transcription from the L strand of fragments Eco RI A, B and D and Hpa II C and that of late transcription from the complementary strands (H strand). We have shown the existence of symmetric transcription within most of fragment Eco RI B and in about one third of fragment Eco RI D. We have also found that sus mutants in cistron 4 do not induce the synthesis of late RNA suggesting that protein p4 is involved in the control of late transcription.

Disruption of the nucleosomes at the replication fork.

The fate of parental nucleosomes during chromatin replication was studied in vitro using in vitro assembled chromatin containing the whole SV40 genome as well as salt‐treated and native SV40 minichromosomes. In vitro assembled minichromosomes were able to replicate efficiently in vitro, when the DNA was preincubated with T‐antigen, a cytosolic S100 extract and three deoxynucleoside triphosphates prior to chromatin assembly, indicating that the origin has to be free of nucleosomes for replication initiation. The chromatin structure of the newly synthesized daughter strands in replicating molecules was analysed by psoralen cross‐linking of the DNA and by micrococcal nuclease digestion. A 5‐ and 10‐fold excess of protein‐free competitor DNA present during minichromosome replication traps the segregating histones. In opposition to published data this suggests that the parental histones remain only loosely or not attached to the DNA in the region of the replication fork. Replication in the putative absence of free histones shows that a subnucleosomal particle is randomly assembled on the daughter strands. The data are compatible with the formation of a H3/H4 tetramer complex under these conditions, supporting the notion that under physiological conditions nucleosome core assembly on the newly synthesized daughter strands occurs by the binding of H2A/H2B dimers to a H3/H4 tetramer complex.

Potato spindle tuber viroid. X. Visualization and size determination by electron microscopy.

Purified potato spindle tuber viroid (PSTV) has been visualized by electron microscopy, using the protein monolayer spreading technique. When PSTV solutions in 4 M sodium acetate were spread onto distilled water, short structures, mostly in compact aggregates, were seen. No such structures could be seen with PSTV preparations that had been incubated with ribonuclease or in control preparations devoid of PSTV. When PSTV solutions in 8 M urea were spread onto distilled water or onto 0.015 M ammonium acetate (pH 8), large numbers of individual short strands were seen. No such strands could be discerned in control preparations or in PSTV preparations treated with ribonuclease. Average length of PSTV molecules was about 500 A. Heating of PSTV samples in 8 M urea for 10 min at 63°, followed by quenching in ice water and spreading, had no discernible effect on the appearance or the mean length of PSTV molecules. When PSTV was heat denatured in the presence of 1.1 M formaldehyde, followed by quenching and spreading onto distilled water, the length of PSTV molecules varied; some molecules, however, were up to 700 A long. To determine whether PSTV is a single- or a double-stranded molecule, PSTV in 8 M urea was mixed with native coliphage T7 DNA, or with heat-denatured, formylated carnation mottle virus RNA (CarMV RNA). PSTV and double-stranded T7 DNA had similar widths, but PSTV was clearly thicker than single-stranded CarMV RNA. Comparison of the lengths of T7 DNA and PSTV, spread in urea, showed that T7 DNA is about 280 times longer than PSTV; the comparison of denatured CarMV RNA with denatured PSTV indicated that CarMV RNA is probably about 17 times longer than PSTV. From these data, we conclude that PSTV is probably a single-stranded RNA with some kind of hairpin-like structure and with a molecular weight in the range of 8.0–9.0 × 104 daltons.

Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation.

In the presence of emetine, an inhibitor of protein synthesis, nascent DNA on forward arms of replication forks in hamster cell lines containing either single or amplified copies of the DHFR gene region was enriched 5‐ to 7‐fold over nascent DNA on retrograde arms. This forward arm bias was observed on both sides of the specific origin of bidirectional DNA replication located 17 kb downstream of the hamster DHFR gene (OBR‐1), consistent with at least 85% of replication forks within this region emanating from OBR‐1. However, the replication fork asymmetry induced by emetine does not result from conservative nucleosome segregation, as previously believed, but from preferentially inhibiting Okazaki fragment synthesis on retrograde arms of forks to produce ‘imbalanced DNA synthesis’. Three lines of evidence support this conclusion. First, the bias existed in long nascent DNA strands prior to nuclease digestion of non‐nucleosomal DNA. Second, the fraction of RNA‐primed Okazaki fragments was rapidly diminished. Third, electron microscopic analysis of SV40 DNA replicating in the presence of emetine revealed forks with single‐stranded DNA on one arm, and nucleosomes randomly distributed to both arms. Thus, as with cycloheximide, nucleosome segregation in the presence of emetine was distributive.

Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin

Trimethylpsoralen was used to crosslink the extrachromosomal ribosomal DNA in nucleoli or nuclei of growing Dictyostelium discoideum cells. The DNA was extracted and was examined by spreading under denaturing conditions for electron microscopy. Intact 95,000 base ribosomal DNA molecules were seen, showing regularly spaced, single-stranded bubbles of about 200 to 400 bases in size, interrupted twice by 11,000 base heavily crosslinked stretches, which correspond to the known positions of the coding regions. The bubbles on the nontranscribed regions indicate the presence of nucleosomes during crosslinking. The DNA was digested with restriction enzymes and analysed by gel electrophoresis in parallel with DNA not treated with psoralen. Fragments from the non-coding region had the same mobility as untreated DNA, while those from the coding region had a markedly lower mobility, though not as low as that of crosslinked pure DNA. This shifting of the bands, specific to the coding region, was also seen when whole cells were treated with psoralen. Treatment of nucleoli with 2 m-NaCl (which is known to dissociate histones) before addition of psoralen led to strong crosslinking all along the ribosomal DNA, resulting in a decreased electrophoretic mobility of bands from the non-coding region, but no further retardation of those from the coding region. In differentiating Dictyostelium cells, slugs, where ribosomal RNA synthesis is very much reduced, the extent of psoralen-crosslinking in the coding region was reduced, but not completely to the level of that of the non-transcribed spacer. In order to test whether psoralen itself alters chromatin structure, crosslinked and non-crosslinked nucleoli from growing cells were lysed with heparin and spread for electron microscopy. There was no difference in the appearance or the frequency of the transcription units seen. Digestion of crosslinked nuclei with micrococcal nuclease indicated an undisturbed structure for bulk chromatin, as well as for the chromatin in the non-transcribed spacer of the ribosomal DNA. Thus psoralen-crosslinking does not lead to extensive disruption or distortion of the structure of either inactive or active chromatin. We conclude, taking the results presented in the Appendix into account, that the extent of psoralen-crosslinking in chromatin DNA is diagnostic for the structure of undistorted chromatin.(ABSTRACT TRUNCATED AT 400 WORDS)