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

Abstract

DNA-protein cross-linkages were formed in intact nuclei of chicken erythrocytes and liver cells by the action of cis-diammine dichloroplatinum (II). Most cross-linked proteins were components of the nuclear matrix, and their heterogeneity reflected the different complexity of liver and erythrocytes matrices, respectively. Some basic proteins, including histones, were also cross-linked, particularly in erythrocyte nuclei. South-Western blotting revealed that a variety of proteins isolated from the cross-linked liver nuclei recognized DNA specifically. In this group of proteins two relatively abundant, acidic, species of 38 and 66 kDa, respectively, might represent novel DNA-binding proteins from the nuclear matrix. In the case of erythrocytes, only the basic proteins showed a DNA-recognition capacity, and among them there were some unidentified species, absent from liver. Lamin B2 was cross-linked but was unable to recognize DNA, and the same was true for other abundant, cross-linked proteins from both types of nuclei. This led to the hypothesis that for some DNA-nuclear matrix interactions the aggregation typical of matrix proteins is essential for the specificity of DNA recognition. Hybridization analysis of the DNA isolated from the cross-linked complexes showed that SARs (scaffold attachment regions) and telomeric sequences were well represented in the cross-linked fragments, that the cross-linked DNA of liver was partially different from that of erythrocytes and that two defined SAR sequences were found to be present only in the cross-linked DNA. These results are in agreement with the present views on DNA-nuclear matrix interactions, which are usually studied on isolated nuclear matrices or purified proteins. Instead, our results provide experimental evidence obtained directly from intact nuclei.

Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome

“…This phenomenon may be due to incomplete binding of the S͞MAR-protein complex to HAP as frequently observed with this technique (27). Both in vitro and in vivo, only the tetrameric, but not the dimeric, S͞MAR construct is recognized by the nuclear matrix protein SAF-A, as shown for pEPI-1 (18).…”

“…Cells were crosslinked with 1 mM cis-diammineplatinum(II)-dichloride and lysed in the appropriate buffer (see Methods). The DNA was then fragmented by sonification, and the matrix-associated DNA-protein complex was adsorbed by HAP, whereas all noncrosslinked material stayed in the supernatant (27). DNA from the supernatant and the HAP-bound material was isolated, and identical amounts of DNA were used for PCR analysis by using primers derived from the CMV promoter (17,18).…”

“…Cells (n ϭ 4 ϫ 10 7 ) were collected by centrifugation, and the cis-platin crosslinking was performed as described by Ferraro and coworkers (26,27). Crosslinking was stopped, and the cells were resuspended in 10 ml of lysis buffer (5 M urea͞2 M guanidine hydrochloride͞2 M NaCl͞1 mM PMSF) and bound to hydroxyapatite (HAP, Bio-Rad).…”

Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteins

“…specifically crosslinked to native AnkA by cisdiamminedichloroplatinum (cisplatin or cis -DDP) (Ferraro et al ., 1996;Jenke et al ., 2002;VanderWaal et al ., 2002). When native AnkA was immunoprecipitated from A. phagocytophilum -infected HL-60 cells, DNA was only coimmunoprecipitated from cis-DDP-treated preparations (Fig.…”

“…To demonstrate the exclusive presence of AnkA in nuclei and to exclude the possibility of contamination with whole A. phagocytophilum, the protein concentration of the nuclear lysates was measured and identical quantities of the same nuclear prepration were loaded for electrophoresis and immunoblotted for the detection of AnkA and Msp2 protein as above. Immunoblots were also used to demonstrate changes in electrophoretic mobility of AnkA after cis-DDP cross-linking or DNase treatments (Ferraro et al, 1996;Jenke et al, 2002;VanderWaal et al, 2002). For immunoblots, alkaline phosphatase-labelled goat anti-rabbit or mouse IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was used as a secondary antibody.…”