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

Abstract

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...

Highly Preferential Linker Histone Binding to Actinomycin D-Treated DNA

“…Many recent studies revealed that, upon binding of ActD, DnA became severely distorted (2,5,13,14). in our recent works we have established that the linker histones possessed the property of binding to irregular DNA structures in a sequence independent manner (7,16,17). Since the bound ACTD modified conformationally the 2073 bp DNA-fragment bearing multiple "perfect" and "imperfect" sites for the drug binding, the drug-enhancing effect on lh/ DNA interactions might be a consequence of the formation of unusual DnA structures attracting the linker histones.…”

“…Since it was demonstrated that lh bound preferentially to unusual (mainly bent, curved, kinked) DnAstructures (7,16,17), the observed stimulating effect of ActD on lh/DnA interactions in competitive reactions could be partially explained: the resulting modified structures might be attractive for the histone binding. intercalation of ActD and the subsequent invasion into the DNA minor groove causes double helix conformational changes and major-groove widening (2,5).…”

“…Formation of histone H1/DNA complexes and drugcompetition assay the complexes of linker histones h1, h5 and GD5 with each of the DnA-fragments (large or small, ActD-treated or intact) were created by direct mixing with 1µg DnA in 15 µl sample mixture containing 20 mM tris-hcl, ph 7.5, 20 mM nacl, 0.1 mM eDtA, 0.1mM phenylmethylsulfonyl fluoride (PMSF), at protein to DNA ratio of 1.0 w/w (15,16,17). Following incubation for 25 min at room temperature (usually 23 o c), 1.5 µl of a loading mix (0.1 % bromophenolblue, 30 % glycerol) was added and the samples electrophoresed on 1.0% agarose gel for the large DnA-fragment and on 15 % polyacrylamide gel (29:1 AA to bisAA) for the small 34 bp fragment.…”

Topological Differences in the Interaction of Human DNA Topoisomerase I with DNA-Histone Complexes Modified by cis- and trans-DDP

“…9) Yaneva and colleagues have previously reported that the major chromatin protein histone H1 binds preferentially to cis-DDP-modified DNA. 10) In this study, we demonstrate that trefoil, figure-eight knot, fragment DNA, and a mini circular closed knot can be obtained in a yield of approximately 30% by the reaction of DNA topo I with cis-DDP-modified fX174DNA-histone complexes. The yield of the mini circular closed knot and trefoil from trans-DDP-modified fX174DNA-histone complexes is only approximately 2%, however.…”

The Non-histone Chromatin Protein HMG1 Protects Linker DNA on the Side Opposite to That Protected by Linker Histones

“…Indeed, early evidence that chromatosome-sized units containing either H1 or HMG1 could be isolated from MNase digests of whole chromatin (18,19) would argue indirectly that the binding of the two classes of linker proteins might be mutually exclusive, and the two proteins might compete with each other for binding to the nucleosome. That the two proteins compete for binding to four-way junction DNA (20,21) or cisplatin-modified DNA (22) has been demonstrated recently. Preliminary experiments in our laboratory show that this may also be the case for reconstituted nucleosomes.…”