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1.
In order to determine the time required for nucleosomes assembled on the daughter strands of replication forks to assume favoured positions with respect to DNA sequence, psoralen cross-linked replication intermediates purified from preparative two-dimensional agarose gels were analysed by exonuclease digestion or primer extension. Analysis of sites of psoralen intercalation revealed that nucleosomes in the yeast Saccharomyces cerevisiae rDNA intergenic spacer are positioned shortly after passage of the replication machinery. Therefore, both the 'old' randomly segregated nucleosomes as well as the 'new' assembled histone octamers rapidly position themselves (within seconds) on the newly replicated DNA strands, suggesting that the positioning of nucleosomes is an initial step in the chromatin maturation process.  相似文献   

2.
Conservative assembly and segregation of nucleosomal histones   总被引:28,自引:0,他引:28  
I M Leffak  R Grainger  H Weintraub 《Cell》1977,12(3):837-845
The assembly of new histones into nucleosomes and the segregation of old histones during replication were investigated using a density gradient, sedimentation equilibrium analysis of histones labeled in vivo with dense amino acids. After a 1 hr pulse of dense amino acids and 3H-lysine, nucleosomes were isolated from chick myoblast organ cultures, and the histones were cross-linked to octamers. The octamers were purified from DNA and then banded to equilibrium in cesium-formate guanidinium-HCI density gradients. The cross-linked dense octamers have the same density as the noncross-linked dense histones, and both were significantly heavier than histones synthesized in the presence of light amino acids. This experiment shows that new histone does not mix with old histone in the new nucleosomes, since the labeling protocol allows density labeling of only one histone for every seven preexisting unlabeled histones. Thus the assembly of new histone octamers is conservative. Using essentially the same experimental design, but varying the details of the labeling procedures, we also show that the dense histone octamer is stable over 3-4 generations, that neighboring octamers tend to be synthesized at the same time, and that old and new histone octamers segregate conservatively over 2-3 generations.  相似文献   

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Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de-novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF-1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.  相似文献   

5.
The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concentration of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.  相似文献   

6.
Stability of the conservative mode of nucleosome assembly.   总被引:7,自引:2,他引:5       下载免费PDF全文
The conservative assembly of nucleosome histone octamer cores has been confirmed by electrophoretic analysis of density labeled histones following equilibrium buoyant density centrifugation. After normal replication, crosslinked octamers are shown not to contain a mixture of new and old core histones. Moreover, when DNA synthesis is inhibited by ara-C nucleosome cores are still assembled exclusively from nascent histone. Similarly, after release from cycloheximide inhibition newly synthesized core histone is conservatively deposited. Thus, a conservative mechanism of histone octamer assembly occurs when nascent histone is present in the normal stoichiometry to nascent DNA and when chromatin is assembled in nascent histone or nascent DNA excess.  相似文献   

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9.
Assembly of active chromatin   总被引:3,自引:0,他引:3  
S Kumar  M Leffak 《Biochemistry》1986,25(8):2055-2060
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10.
The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described. All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present. Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion. This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present. Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained. These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants. This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation. The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not. Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion.  相似文献   

11.
The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.  相似文献   

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14.
Previous work has shown that DNA and the histone proteins will combine to form structures of a complex, yet definite nature. Here, we describe three experiments aimed at a better understanding of the interactions of DNA with the histone octamer and with histone H5. First, there has been some question as to whether the methylation of DNA could influence its folding about the histone octamer. To address this point, we reconstituted the histone octamer onto a 440 base-pair DNA of defined sequence at various levels of cytosine methylation, and also onto the unmethylated DNA. The reconstituted structures were probed by digestion with two different enzymes, micrococcal nuclease and DNase I. All samples were found to contain what appear to be three histone octamers, bound in close proximity on the 440 base-pair DNA. The cutting patterns of micrococcal nuclease and DNase I remain the same in all cases, even if the DNA has been extensively methylated. The results show, therefore, that methylation has little, or no, influence on the folding of this particular DNA about the histone octamer. Second, there has been concern as to whether the base sequence of DNA could determine its folding in a long molecule containing several nucleosomes, just as it does within any single, isolated nucleosome core. In order to deal with this problem, we cut the 440 base-pair DNA into three short fragments, each of nucleosomal length; we reconstituted each separately with the histone octamer; and then we digested the reconstituted complexes with DNase I for comparison with similar data from the intact 440 base-pair molecule. The results show that the folding of this DNA is influenced strongly by its base sequence, both in the three short fragments and in the long molecule. The rotational setting of the DNA within each of the three short fragments is as predicted from a computer algorithm, which measures its homology to 177 known examples of nucleosome core DNA. The rotational setting of the DNA in the 440 base-pair molecule remains the same as in two of the three short fragments, but changes slightly in a third case, apparently because of steric requirements when the nucleosomes pack closely against one another. Finally, there has been little direct evidence of where histone H5 binds within a DNA-octamer complex.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

15.
Attraction, phasing and neighbour effects of histone octamers on curved DNA   总被引:6,自引:0,他引:6  
Nucleosome core particles were reconstituted on various DNA fragments containing a Crithidia fasciculata kinetoplast curved tract. The results show that, on curved DNA, nucleosome core particles form six- to sevenfold preferentially, relative to bulk sequences. The preferential deposition occurs at multiple periodic positions, whose distribution reveals a unique rotational setting of DNA with respect to the histone octamer surface and whose average periodicity is 10.26 +/- 0.04. Evidence is provided for a context effect in histone octamer deposition: octamers bound to a segment of curved DNA influence the positions of neighbour octamers. Taken together, the preferential formation of nucleosome core particles and the influence on the localization of neighbouring particles suggest for intrinsically bent sequences the biologically relevant role of organizers of nucleosomal arrays.  相似文献   

16.
A major question in chromatin involves the exact organization of nucleosomes within the 30-nm chromatin fiber and its structural determinants of assembly. Here we investigate the structure of histone octamer helical tubes via the method of iterative helical real-space reconstruction. Accurate placement of the x-ray structure of the histone octamer within the reconstructed density yields a pseudoatomic model for the entire helix, and allows precise identification of molecular interactions between neighboring octamers. One such interaction that would not be obscured by DNA in the nucleosome consists of a twofold symmetric four-helix bundle formed between pairs of H2B-α3 and H2B-αC helices of neighboring octamers. We believe that this interface can act as an internucleosomal four-helix bundle within the context of the chromatin fiber. The potential relevance of this interface in the folding of the 30-nm chromatin fiber is discussed.  相似文献   

17.
18.
The preparation of hybrid histone octamers with wheat histone H2A variants replacing chicken H2A in the chicken octamer is described. The fidelity of the reconstituted hybrid octamers was confirmed by dimethyl suberimidate cross-linking. Polyglutamic-acid-mediated assembly of these octamers on long DNA and subsequent micrococcal nuclease (MNase) digestion demonstrated that, whereas chicken octamers protected 167 base-pairs (representing 2 full turns of DNA), hybrid histone octamers containing wheat histone H2A(1) with its 19 amino acid residue C-terminal extension protected an additional 16 base pairs of DNA against nuclease digestion. The protection observed by hybrid histone octamers containing wheat histone H2A(3) with both a 15 residue N-terminal and a 19 residue C-terminal extension was identical with that observed with H2A(1)-containing hybrid histone octamers with only the 19 residue C-terminal extension. These results suggest that the role of the C-terminal extension is to bind to DNA of the "linker" region. The thermal denaturation of chicken and hybrid core particles was identical in 10 mM-Tris.HCl.20 mM-NaCl, 0.1 mM-EDTA, confirming that there was no interaction between the basic C-terminal extension and DNA of the core particle. Denaturation in EDTA, however, showed that hybrid core particles had enhanced stability, suggesting that the known conformational change of core particles at very low ionic strength allows the C-terminal extension to bind to core particle DNA under these conditions. A model accounting for the observed MNase protection is presented.  相似文献   

19.
Recently, we have found that the assembly of nucleosomes reconstituted on negatively supercoiled DNA is cooperative. In the present paper the role of DNA topology and of histone tails in nucleosome assembly was explored. Reconstituted minichromosomes on relaxed DNA at different histone/DNA ratios (R) were assayed by topological analysis and electron microscopy visualization. Both methods show a linear relationship between average nucleosome number (N) and R. This suggests that in the case of relaxed DNA, cooperative internucleosomal interactions are small or absent. The influence of histone tails in nucleosome assembly was studied on minichromosomes reconstituted with trypsinized histone octamer on negatively supercoiled DNA by topological analysis. The topoisomers distribution, after trypsinization, dramatically changes, indicating that nucleosome-nucleosome interactions are remarkably decreased. These results show that, in chromatin folding, in addition to the well known role of histone H1, the interactions between histone octamer tails and DNA are also of importance.  相似文献   

20.
Unwinding of chromatin by the SV40 large T antigen DNA helicase.   总被引:6,自引:1,他引:5       下载免费PDF全文
U Ramsperger  H Stahl 《The EMBO journal》1995,14(13):3215-3225
We have analysed the unwinding of nucleosomally organized DNA by simian virus 40 large tumour (T) antigen. Isolated T antigen can bind to existing nucleosome cores containing the viral replication origin sequence, which results in displacement of the histone octamer and unwinding of the DNA. However, specific binding to nucleosome cores is salt sensitive and nearly completely blocked under ionic conditions that otherwise support DNA replication. Once started, the progressing T antigen helicase, like an elongating RNA polymerase, is not further repressed by histone octamers, irrespective of the presence or absence of linker histone H1. Disruption of the nucleosomal structure in the process of unwinding may be assisted by the demonstrated interaction of the hexameric T antigen complex with histone proteins H1 and H3. Finally, our studies reveal the inability of topoisomerase I and/or II to continually relieve the superhelical tension of covalently closed circular minichromosomes as generated during their unwinding by T antigen. This may indicate that chromatin relaxation during the process of DNA replication can only be efficiently performed by a topoisomerase that is (trans)activated by other factors.  相似文献   

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