Purification of histone H10 and its subfractions under non-denaturing conditions

Banchev, Todor; Srebreva, Ljuba; Zlatonova, Jordanka

doi:10.1016/0304-4165(91)90208-x

Cited by 19 publications

(10 citation statements)

References 12 publications

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“…Chicken erythrocyte histone H1 was obtained under nondenaturing conditions (10) and checked for purity by SDS-containing polyacrylamide gel electrophoresis (11). The globular domain of histone H1 was prepared as outlined in Krylov et al (3).…”

Section: Methodsmentioning

confidence: 99%

H1 Binding Unwinds

Ivanchenko

Hassan

Holde

et al. 1996

Journal of Biological Chemistry

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The preference of the linker histones to bind to superhelical DNA in comparison with linear or relaxed molecules suggests that these proteins might, in turn, change the twist and/or writhe of DNA molecules upon binding. In order to explore such a possibility, we looked for changes in the linking number of plasmid pBR322 caused by H1 binding, using assays that involve nicking and resealing of DNA strands. Two types of enzymes were used, eukaryotic topoisomerase I and prokaryotic DNA ligase. The results revealed that H1 binding causes unwinding of the DNA, with the unwinding angle being approximately 10°. The globular domain of histone H1 is also capable of unwinding DNA, but to a lesser degree.It has been known for a number of years that the linker histones (H1, H5, and their variants) exhibit a preference to bind to superhelical DNA over linear or relaxed DNA (1-3). A reasonable corollary to this observation is that these proteins, in binding to superhelical DNA, might change the twist of the double helix and/or the writhing of the helical axis in space. Consistent with this notion, we have recently shown that titration of superhelical plasmid DNA with increasing amounts of histone H1 changes the patterns of cleavage by single strandspecific nucleases, causing disappearance of preexisting nuclease-sensitive sites at low and moderate H1 levels, followed by the appearance of new sites at high protein levels (4). A possible explanation for the disappearance of preexisting sites is that histone H1 binding absorbs some of the negative superhelical stress in the molecule (unwinds the DNA), so that the decreased stress leads to loss of stress-dependent sensitive sites.Previous work on the binding of histone H1 or H5 to superhelical plasmids has led to contradictory interpretations. BinaStein and Singer (5) interpreted their data as indicating stabilization of preexisting superhelical turns by H1 binding, with no new superhelical turns being created. Stein (6) and Morse and Cantor (7), on the other hand, did not observe stabilization of preexisting superhelical turns. More recently, Sheflin et al. (8) reported that histone H1 was not able to change the linking number of superhelical DNA when assayed in the topoisomerase I-mediated relaxation assay (see below).What is required is a very careful analysis using more than one technique, since the effects might well be small. We decided to utilize both the topoisomerase I-mediated relaxation assay and the ligase-mediated supercoiling assay. The results indicate that linker histones binding unwinds DNA. EXPERIMENTAL PROCEDURESPreparation of Plasmid DNA and Histone H1-Plasmid pBR322 was prepared by CsCl purification and phenol extraction (9). Chicken erythrocyte histone H1 was obtained under nondenaturing conditions (10) and checked for purity by SDS-containing polyacrylamide gel electrophoresis (11). The globular domain of histone H1 was prepared as outlined in Krylov et al. (3). The concentration of the protein stock solutions was estimated by scanning of Coomassie-stained...

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Section: Methodsmentioning

confidence: 99%

H1 Binding Unwinds

Ivanchenko

Hassan

Holde

et al. 1996

Journal of Biological Chemistry

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“…For comparative purposes, human placenta H1 histones were also isolated by extraction with 0.5 M NaCl as described previously (26). To remove the low mobility group proteins, NaCl extract was treated with trichloroacetic acid (2%, w/v).…”

Section: Methodsmentioning

confidence: 99%

The Microheterogeneity of the Mammalian H10Histone

Lindner

Sarg

Hoertnagl

et al. 1998

Journal of Biological Chemistry

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The linker histones H1, H5, and H1 0 are associated with the core histone-DNA complex and with the linker DNA between adjacent nucleosomes and are thought to modulate the condensation/decondensation of the chromatin fiber, thus influencing many nuclear activities such as transcription, replication, recombination, and DNA repair (2). H1 0 was first described in 1969 by Panyim and Chalkley (3, 4) as an H1-like protein present in mammalian tissues with little or no cellular proliferation and was later shown to increase at a terminal stage of differentiation (5-10). Some cells, however, accumulate significant amounts of the protein while still actively proliferating (11, 12) or accumulate it upon proliferation arrest without concomitant differentiation (12). In addition, H1 0 seems to be the only histone undergoing changes during malignant transformation (13). Recently, it was found that transformation of NIH 3T3 fibroblasts by c-Ha-ras Val12 oncogene causes chromatin decondensation accompanied by alterations in the content of histone H1 0 (14). All these findings suggest a role for H1 0 in the regulation of either cell proliferation or cellular differentiation.In every tissue in which H1 0 has been detected, two subfractions were present (15-18). It appears that these two H1 0 proteins, up to now named H1 0 a and H1 0 b, have specific individual functions in chromatin (15). The relative proportions of the two H1 0 forms seem to differ from tissue to tissue (15) and exhibit age-dependent changes in rat brain cortical neurons (17). The two H1 0 s are resolvable by ion-exchange chromatography on Bio-Rex 70 (16, 19) or acetic acid-urea gel electrophoresis (15-18). Most recently, Lindner et al. (20) developed a high performance capillary electrophoresis method allowing separation of H1 0 and its subfractions from other histone H1 subtypes. The two H1 0 proteins run coincidentally on sodium dodecyl sulfate-polyacrylamide gels, suggesting that the difference between them is one of charge and not of size (15). Since neither treatment with alkaline phosphatase nor exposure to alkaline conditions changed the separation of the H1 0 peak into two subfractions, phosphorylation and ADP-ribosylation have been ruled out as possible post-translational modifications responsible for the different forms (16,20,21). Although some investigators speculated that the two forms of H1 0 might be coded by different genes (15, 17), Doenecke et al. (22) found that the mammalian genomes contain only one H1 0 gene.To gain insight into the nature of the two H1 0 subfractions, we took advantage of a combined reversed phase high performance liquid chromatography (RP-HPLC) 1 /hydrophilic interaction liquid chromatography (HILIC) technique recently developed in our laboratory for separating acetylated core and phosphorylated H1 histones (23,24). By applying this two-step HPLC method human placenta histone H1 0 was resolved into four components, which were treated with cyanogen bromide and chymotrypsin. HILIC analysis of the peptide fragments

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“…Histone H1 was isolated from mouse liver nuclei under nondenaturing conditions on CM Sephadex C25 columns (23). HMG1 from chicken erythrocytes was obtained from a 0.5 M NaCl extract of crude nuclei by chromatography on a Polybuffer-exchange column (PBE94, Pharmacia) according to ref.…”

Section: Methodsmentioning

confidence: 99%

The major chromatin protein histone H1 binds preferentially to cis -platinum-damaged DNA

Yaneva

Leuba

Holde

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Both cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) and trans-diamminedichloroplatinum (II) form covalent adducts with DNA. However, only the cis isomer is a potent anticancer agent. It has been postulated that the selective action of cis-DDP occurs through specific binding of nuclear proteins to cis-DDP-damaged DNA sites and that binding blocks DNA repair. We find that a very abundant nuclear protein, the linker histone H1, binds much more strongly to cis-platinated DNA than to trans-platinated or unmodified DNA. In competition experiments, H1 is shown to bind much more strongly than HMG1, which had been previously considered a major candidate for such binding in vivo.cis-Diamminedichloroplatinum(II) (cisplatin or cis-DDP) is a potent chemotherapeutic agent widely used in the clinical practice to treat several types of human malignancies. The therapeutic effect is believed to arise as a consequence of cis-DDP binding to DNA (1), but it cannot be solely explained on the basis of DNA binding because a number of closely related compounds, among which is included the geometric isomer trans-DDP, are not effective agents although they also damage DNA. The differential biological effect of different Pt compounds may lie in the differential processing of different Pt-DNA adducts by the cell. The realization that different adducts may be processed differently suggested that certain proteins might enhance or block DNA repair by specifically interacting with cis-DDP-modified DNA; this has focused research toward identifying such proteins.cis-DDP causes the formation of two major intrastrand DNA adducts, 1,2-d(GpG) and d(ApG) cross-links in which the two chloride ions of cis-DDP are replaced by the N7 atoms of guanine and adenine. An intrastrand cross-link may also be formed, at a much lower frequency, at d(GpXpG), where X is any base. Other minor adducts may also arise, including interstrand cross-links involving guanine residues on opposite strands. Biochemical and structural analyses of both intra-and interstrand cis-DDP adducts (2, 3) reveal major distortions of the DNA double helix, including bending and unwinding (for review, see ref. 4). The therapeutically inactive trans-DDP is incapable of forming the 1,2-(GpG) and d(ApG) adducts for stereochemical reasons. It does form 1,3-intrastrand links and also cross-links opposite strands, but although cis-DDP reacts with guanine residues in d(GpC), the trans-isomer preferentially cross-links complementary guanine and cytosine residues (5). The different kinds of cross-links created by the cis-and trans-DDP are illustrated schematically for one specific DNA fragment used in this work (see Fig. 2 A).Recent years have witnessed the discovery of a number of cellular proteins, mostly with still unidentified in vivo functions, that recognize and bind selectively to cis-DDP-modified DNA. These include the relatively abundant chromatin nonhistone proteins HMG1 and HMG2 (6, 7), the human structure-specific recognition protein 1 SSRP1 (8, 9), the yeast intrastrand c...

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Purification of histone H10 and its subfractions under non-denaturing conditions

Cited by 19 publications

References 12 publications

H1 Binding Unwinds

H1 Binding Unwinds

The Microheterogeneity of the Mammalian H10Histone

The major chromatin protein histone H1 binds preferentially to cis -platinum-damaged DNA

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