Histone cross-linking

Similar results were obtained from reconstitution experiments with DNA and a non-cross-linked octamer (Thomas and Butler, 1978). Nucleosome-like particles were observed in the EM and a pattern of histone cross-linking comparable to that of native chromatin was obtained. However, only 140-base-pair repeats were obtained upon micrococcal nuclease digestion instead of 200-base-pair repeats obtained for native rat liver chromatin (Noll and Komberg, 1977). This indicates that, in the absence of HI, only core particles can be reconstituted. Nevertheless, these studies with both cross-linked and reassembled un-cross-linked histones demonstrate that the octamer is a complete biological functional unit retaining the information for folding the DNA around the histone core. 

Reale A, Malar M, Zaido G et al. In vitro induction of Hl-Hl histone cross-linking by adenosine diphosphate-ribose polymers. Biochemistry 2000 39 10413-10418. 

It has been found that DTBP cross-linking substantially increased the salt stability of the complexes. The salt stabilization is reversed upon the addition of DTT, which cleaves the bifunctional reagent, indicating that it is not due to the conversion of the amines to amidines and is dependent upon the cross-linking. Similar results were achieved with other polycations, including poly(allylamine), and histone HI. 

Jackson V. Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent. Cell 1978 15 945-954. 

Nickel chloride has been reported to induce DNA strand breaks in CHO cells [435] in a concentration, which did not significantly injure normal cellular division, and DNA-protein cross-links, which were concentration- and time-dependent and preferentially occurred in cells in the late S phase of the cell cycle [436], The nickel cross-linked proteins included nonhistone chromatin proteins, nonhistone DNA-binding proteins and a 30 kDa protein that comigrated electrophoretically with histone HI. Moreover, blocking of cell growth in S phase [249] and induction of DNA repair synthesis in CHO cells [437] and reduction in the fidelity of DNA synthesis [438, 439], have been reported. 

Huang X, Okafuji M, Traganos F, Luther E, Holden E, Darzynkiewicz Z. Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin. Cytometry A. 2004 Apr 58(2) 99-110. 

Identification of Cross-Linked Histone Dimers and Trimers... 

Specifically, when histones are dissociated from DNA in 2 M NaCl, and H3 H4 tetramer and H2A-H2B dimer may be identified after fractionation of the histones at pH 5.0 and cross-linking with dimethylsu-berimidate (Komberg and Thomas, 1974). The inference was drawn that these complexes exist as such in the intact nucleosome. Furthermore, since both the un-cross-linked H3 H4 tetramer, and the uncross-linked H2A-H2B dimer are stable complexes, it has proved possible to characterize their physical properties in solution. Some of these results are summarized here. 

Cross-linking of chromatin in 2 M NaCl at pH 8.0 results in the formation of a cross-linked octamer (Thomas and Komberg, 1975a) which contains the four core histones in equimolar ratios (Thomas and Komberg, 1975b). A non-cross-linked complex of histones isolated in 2 M NaCl at pH 7.0 also was found to contain an equimolar ratio of the four core histones but had a molecular weight determined to be near that... 

The histone octamer of nucleosome core particles was cross-linked by dimethylsuberimidate and isolated from the DNA by precipitation in 3 M NaCl (0.05 M sodium phosphate buffer, pH 7.0). The cross-linked octamer, dissolved at low ionic strength, was reconstituted by mixing with DNA at 1.0 M NaCl (pH 8.0 Tris buffer) and dialyzed against 0.6 M NaCl in the same buffer. The reconstituted particle had properties similar to those of the cross-linked core particle. It sedi-... 

As described in Section II,B, an H2A H2B-H4 trimer may be obtained by UV cross-linking of chromatin. Therefore, H2B apparently has two distinct binding sites for H2A and H4 (Martinson et al., 1979a). The lack of trimer formation by addition of a third histone to a preformed pair consequently must indicate that a trimeric unit is not stable. The dimer is the basic structural unit in the assembly of histones (Sperling and Bustin, 1976). This idea is supported by the fact that in a mixture of histones only even complexes have been isolated,... 

When the four core histones are mixed in equimolar ratio at low ionic strength a single sedimentation velocity peak is observed with Sjo.w = 2.0. Gel filtration and cross-linking with dimethylsuberimi-date showed that the peak contained a mixture of dimers (Sperling... 

Although the quantitation of the cross-linking data is limited, as discussed above, comparison of the cross-linked histones obtained from chromatin with histone complexes obtained in the absence of DNA can help in developing a more detailed picture of histone-histone interactions. An H2A-H2B dimer was obtained by cross-linking of chromatin with short-range cross-linkers (for references, see Section... 

The histone octamer is the histone unit of the nucleosome. As discussed in Section II, it has been shown that at high salt concentration (7 > 2 M) the core histones can assemble on their own, in the absence of DNA, to form histone octamers (this assembly occurs with both acid- and salt-extracted histones). Furthermore, the secondary and tertiary structures of core histones at high salt concentration are similar to the structures they have in the intact nucleosome. The basic units of the assembly of the four core histones are histone dimers which are obtained at low salt concentration. Upon increase in salt concentration, tetramers, hexamers, and octamers are obtained. The cross-linking pattern of histones in high salt concentration is similar to that in chromatin, again supporting the idea that the assembly of core histones at high salt concentration is similar to that in chromatin. 

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