Crosslinking of nuclear proteins to DNA by cis‐diammminedichloroplatinum in intact cells Involvement of nuclear matrix proteins

Abstract

In order to detect the nuclear matrix proteins involved in DNA binding, avoiding possible artifacts derived from the disruption of nuclei. proteins were crosslinkcd to DNA by the action of cis-diammincdichloroplatinum on intact chicken liver cells and analyzed by twodimcnsional gel eleetrophoresis. At Icast eleven spccics ofcrosslinkcd proteins wcrc found to dcrivc from the nuclear matrix prepared from the same cell type, and live of these were found also among the proteins crosslinked to DNA in intact liver cells from ox and pig This subset ofcommon proteins, conserved in different animal species. is likely to have a fundamental role for the anchorage of DNA to the nuclear matrix.DNA: Nuclear protein; Nuclear matrix: cis-Diamminedichloroplatinum 1, INTRODUCTIONDNA in eukaryotic chromatin is organized into large loops, which are associated with histones and non-histone proteins, and anchored to the nuclear periphery and to an internal proteinaceous structural meshwork called the nuclear matrix or scaffold [l-3]. This is thought to be involved in processes such as DNA replication, t*:anscription, RNA splicing and transport (for reviews see Georgiev e:. al. [4] and Berezney [S]).To better understand both the structural and functional aspects of loops organization it is necessary to identify the DNA-protein interact!ons which occur in the loops-matrix anchoring points. A number of authors have described the scaffold-or matrix-attachment regions (SAR or MAR) of DNA and some of the proteins which appear to be involved in these interactions (reviewed in [6-83). A possible criticism of these results is that they have been obtained after introducing some modification in the native structure of the nuclei. Many different preparations of the nuclear matrix have been proposed, and possibly no one is immune from drawbacks [9]. Thus, for example, extraction of nuclei with high salt may cause a sliding of DNA over its attachment points or an exchange of the DNA-binding proteins, so that the original DNA-protein interactions are altered or lost. AWx!~~iuriotts: c/s-DDP, ris-diamminedicbloroplatinum (II); IEF, isoclectrofocusingCorreqon&ncc uddwss: C. Turano, Department of Biochemical Sciences, University La Sapien=, P. Aldo Moro. 00185 Rome, Italy.Mnilica and coworkers [ 10, 1 l] have demonstrated that heavy metals or their complexes are able to diffuse inside intact cells and nuclei and to crosslink DNA to proteins located nearby. On the basis of an immunoblotting staining an indication was provided that nuclear matrix proteins could be crosslinked to DNA by the use of this technique. Considering the interest in determining the proximity of DNA to proteins without disruption of the nuclear structure, we analyzed in greater detail the proteins crosslinked to DNA by cisdiamminedichloroplatinum II (cis-DDP) in intact, viable cells from mammalian and avian liver, with the aim of identifying the nuclear matrix components among the crosslinked proteins. EXPERIMENTALThe hepatic parenchymal cells preparation from chicken, pi& and ox,...

Evidence that protein disulfide isomerase (PDI) is involved in DNA–nuclear matrix anchoring

“…The observation that PDI co-isolates with the NM and crosslinks to DNA (data not shown) is consistent with earlier reports [Ferraro et al, 1992[Ferraro et al, , 1996 that an isoform of PDI, ERp60, is part of the internal NM. Herein, we show that the presence of 1 mM DTT, presumably through its affects on NM-associated proteins containing redox sensitive disulfide linkages, e.g., PDI, altered the anchoring of DNA loops such that the unwinding of supercoils was enhanced and the rewinding of them was inhibited.…”

“…This notion is consistent with the observation that heat-induced binding of proteins to the NM masks the DTT induced loosening of the DNA-NM anchor complex. Although the exact structure of the complex remains elusive, these results and those of others [Altieri et al, 1993;Ferraro et al, 1992Ferraro et al, , 1996, provide significant new insight into DNA-NM anchoring.…”

Comparison of DNA-protein interactions in intact nuclei from avian liver and erythrocytes: A cross-linking study

“…Wedrychowski et al [1986, 19891 and ourselves [Ferraro et al, 1992 have demonstrated that many proteins cross-linked in such a way in intact cells or nuclei derive from the nuclear matrix.…”

DNA sequences acting as binding sites for NM23/NDPK proteins in melanoma M14 cells

“…Although cis-DDP can also produce proteinprotein cross-linking, an indirect bond mediated by unknown protein(s) seems unlikely in our in vivo cross-linking conditions [Banjar et al, 1985;Ferraro et al, 1992]. Moreover, some of the sequences found in the coimmunoprecipitated DNA were already shown to bind NM23/NPDK by in vitro EMSA experiments [Postel et al, 2000].…”

“…After 60 min of incubation at 378C, 1/200 volume of 1 M thiourea solution was added to stop the cross-linking reaction [Ferraro et al, 1992;Cervoni et al, 2003]. The cells were harvested by centrifugation at 2,000g for 5 min at 48C.…”