The interaction of cisplatin and analogues with DNA in reconstituted chromatin

The influence of chromatin structure on cis-diamminedichloroplatinum(II) (cisplatin) DNA damage was investigated in a reconstituted nucleosome system. Nucleosomes were reconstituted on the somatic 5S rRNA gene from Xenopus borealis using the octamer transfer method of reconstitution. Footprinting techniques, utilising bleomycin and DNase I as the damaging agents, were employed to establish the precise location of positioned nucleosomes with respect to the DNA sequence. Reconstituted nucleosomal DNA was treated with cisplatin and drug-induced DNA adduct formation was quantitatively analysed with a polymerase stop assay using Taq DNA polymerase. A densitometric comparison of the relative damage band intensities between purified and reconstituted DNA revealed regions of relative protection corresponding to the sites of the positioned nucleosome cores. This indicated that the preferred site of cisplatin DNA binding was in the linker region of the nucleosome. Statistical analysis showed significant protection from cisplatin DNA damage in the core region of the nucleosome. Three cisplatin analogues were also investigated in this reconstituted nucleosome system. These analogues, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin), cis-dichlorobis(cyclohexylamine)platinum(II) (cis-[PtCl(2)(C(6)H(11)NH(2))(2)]) and dichloro(N-[3-[(2-aminoethyl)-amino]propyl]acridine-4-carboxamide)platinum(II) (ac-PtenCl(2)(n3)), were also found to target the linker region of the nucleosome. The latter DNA-targeted acridine-platinum complex gave rise to the most predominant footprints of all the Pt compounds tested.     

Use of RNA polymerase as an enzymatic probe of nucleosomal structure.

Nucleosomes prepared from human placental nuclei and Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase EC.2.7.7.6) form stable initiation complexes. This property is utilized as a probe of nucleosome structure. RNA polymerase initiation has been studied on purified nucleosomes, nucleosome cores, and nucleosomal DNA. The affinity of E. coli RNA polymerase for both nucleosome cores and monomers was 5-6 fold less than found for nucleosomal DNA. No difference in apparent initiation Km was found between cores and mononucleosomes. This suggests that initiation does not preferentially occur on the DNA tails of nucleosomes. Once initiated and allowed to form nascent RNA, these complexes are very stable to ionic strength changes. Under conditions in which free enzyme is inactivated with rifampicin, the enzyme in the complex retains activity as demonstrated by its ability to transcribe and reinitiate on both nucleosomes and free DNA. These complexes can be well resolved from free nucleosomes on preparative polyacrylamide gels and both can be eluted from gels for analysis of proteins and DNA sequence complexity. Studies using (125I) labelled nucleosomes show that histones are retained in the initiation complex, and are not dissociated by the enzyme during initiation.     

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.