Proteins tightly bound to HeLa cell DNA at nuclear matrix attachment sites.

Abstract

DNA-protein complexes have been isolated from HeLa cell nuclei and nuclear matrix preparations. Two proteins, 55 and 66 kilodaltons in size, remain bound to HeLa DNA after treatment at 80°C in 2% sodium dodecyl sulfate and purification by exclusion chromatography on Sepharose 2B-CL in the presence of 0.3% sodium dodecyl sulfate. These proteins appear to be tightly bound but not covalently linked to the DNA, and they are distributed over the DNA with an average spacing of 40 kilobase pairs. This spacing distribution remains essentially constant throughout the cell cycle. The proteins are bound to the residual 2% of HeLa cell DNA which remains attached to the nuclear matrix after extensive nuclease digestion, a condition which reduces the average size of the DNA to -150 base pairs. Our results suggest that these tightly bound proteins are involved in anchoring cellular DNA to the nuclear matrix. These tightly bound proteins are identical by partial peptide mapping to proteins found tightly bound to the DNA of mammalian, plant, and bacterial cells (D. Werner and C. Petzelt, J. Mol. Biol. 150:297-302, 1981), implying that these proteins are involved in the organization of chromosomal domains and are highly conserved in both procaryotic and eucaryotic cells.The DNA of eucaryotic cells is organized into structural domains 30 to 90 kilobases (kb) in size. These domains are seen in metaphase chromosomes as loops or rosettes of DNA bound to a protein scaffold (10,40,43). In interphase nuclei the DNA is organized in domains of supercoiling measured at 84 to 94 kb (4,22,57), and the DNA is attached to the nuclear matrix with an average spacing of between 35 and 60 kb (25,48). Cellular DNA replication also takes place in association with the nuclear matrix (16,34,42,58), and eucaryotic DNA replicons have a size distribution similar to that of the structural domains described above (18). Several proteins that are tightly bound to cellular DNA have been implicated in maintaining supercoiled domains, binding DNA to chromosome and nuclear scaffolds, or binding to transcriptionally active genes (3,24,28,37,46,47 1981). However, proteins that mediate the interaction between cellular DNA and the nuclear matrix have not been defined.Using methods initially developed to study the adenovirus terminal protein-DNA complex (14, 21, 50), we isolated a stable DNA-protein complex from HeLa cells that contains two proteins, 55 and 66 kilodaltons (kd) in size. These proteins are tightly bound to cellular DNA and exhibit a constant average spacing of about 40 kb throughout the cell cycle. They are specifically enriched in the nuclear matrix fraction, even after 98% of the cellular DNA is removed by nuclease digestion and the size of the bulk DNA is reduced to approximately 150 base pairs (bp). These proteins also possess peptide map homology to proteins previously reported to be bound to a wide variety of procaryotic, eucaryotic, and plant cells DNAs, even after alkali treatment, pronase digestion, and phenol extraction (7,28,(60)(61)(62)(63...

DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D

“…One potential clue is provided by the fact that C1D binds to DNA in an extremely stable fashion, as evidenced by the fact that it remains DNA-associated even after rigorous extraction procedures. Highly stable nonhistone proteins that are bound to DNA tightly have been identified in various systems, including insects, plants, and mammalian cells (Krauth and Werner 1979;Capesius et al 1980;Bodnar et al 1983;Avramova and Tsanev 1987). Although the biological functions for these proteins are still largely obscure, it is noteworthy that some have been reported to be associated with highly repetitive DNA sequences and to be involved in targeting a subset of genomic DNA to the nuclear matrix (Neuer and Werner 1985;NeuerNitsche et al 1988;Werner and Neuer-Nitsche 1989;Pfutz et al 1992).…”

Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo.

“…The overall alkylation level in rat mitochondrial DNA ex- posed to methylating N-nitroso compounds (N,N-dimethylnitrosamine, N-methyl-N-nitrosourea) exceeds that of rat nuclear DNA by a factor of s5 (Wunderlich et al, 1971;Wilkie and Evans, 1982 The fact that clustered O6-EtdGuo sites were only observed in DNA fragments contained in a fraction of DNA isolated by virtue of its association with specific polypeptides may be explained by two alternative hypotheses. (i) The tightly bound polypeptides found in chromosomal DNA (Bodnar et al, 1983;Krauth and Werner, 1979;Neuer et al, 1983;Werner et al, 1981a) may be involved in a nuclear structure and/or function which renders the vicinal DNA more accessible to the ethyldiazonium ions generated from EtNU (see, e.g., the putative mechanisms a -d mentioned above). (ii) It may be argued that by virtue of particular structural properties of its own, a small fraction of DNA might be more sensitive to ethylation by EtNU.…”

“…As previously shown, highly purified chromosomal DNA still contains distinct types of polypeptides (Bodnar et al, 1983;Krauth and Werner, 1979;Werner et al, 1981a) which are covalently bound to internal ends of DNA molecules (Neuer et al, 1983). DNA fragments containing such DNA-protein complexes may be essential constituents of higher order chromatin structures and/or be involved in the anchorage of DNA in nuclear structures (Bodnar et al, 1983;Werner et al, 1980Werner et al, , 1981a.…”

“…DNA fragments containing such DNA-protein complexes may be essential constituents of higher order chromatin structures and/or be involved in the anchorage of DNA in nuclear structures (Bodnar et al, 1983;Werner et al, 1980Werner et al, , 1981a. Since DNAprotein complexes of this type can be selectively enriched on glass fiber filters (Bodnar et al, 1983;Spiess et al, 1982), the frequency of a specific carcinogen-induced structural modification in the DNA of these complexes can be studied in comparison with the remaining DNA. Using a high-affinity anti-(06-EtdGuo) monoclonal antibody (ER-6;Rajewsky et al, 1980;Adamkiewicz et al, 1982) (Bodnar et al, 1983;Spiess et al, 1982).…”

“…Since DNAprotein complexes of this type can be selectively enriched on glass fiber filters (Bodnar et al, 1983;Spiess et al, 1982), the frequency of a specific carcinogen-induced structural modification in the DNA of these complexes can be studied in comparison with the remaining DNA. Using a high-affinity anti-(06-EtdGuo) monoclonal antibody (ER-6;Rajewsky et al, 1980;Adamkiewicz et al, 1982) (Bodnar et al, 1983;Spiess et al, 1982). The material specifically adsorbed to the filters was recovered and, in parallel with equal amounts of the material that had passed through the filters, was radiolabeled with 1251I exclusively on tyrosines (Neuer et al, 1983).…”