We identified four proteins in nuclear extracts from HeLa cells which specifically bind to a scaffold attachment region (SAR) element from the human genome. Of these four proteins, SAF‐A (scaffold attachment factor A), shows the highest affinity for several homologous and heterologous SAR elements from vertebrate cells. SAF‐A is an abundant nuclear protein and a constituent of the nuclear matrix and scaffold. The homogeneously purified protein is a novel double stranded DNA binding protein with an apparent molecular weight of 120 kDa. SAF‐A binds at multiple sites to the human SAR element; competition studies with synthetic polynucleotides indicate that these sites most probably reside in the multitude of A/T‐stretches which are distributed throughout this element. In addition we show by electron microscopy that the protein forms large aggregates and mediates the formation of looped DNA structures.
We show that SAF-A, a nuclear protein which specifically binds vertebrate scaffold-attachmentregion (SAR) elements with high affinity is identical with hnRNP-U, assumed to be involved in packaging of hnRNA in ribonucleoprotein particles. Ultraviolet cross-linking experiments show that the protein, referred to as hnRNP-U/SAF-A, is bound to chromosomal DNA in vivo. Zn vitro, the isolated protein binds to double-stranded and single-stranded DNA and forms higher ordered nucleic-acid -protein complexes. Filter-binding experiments performed with different types of natural and synthetic nucleic acids as substrates show that the protein binds DNA and RNA with different affinities and most likely at different binding sites. We conclude that hnRNP-U/SAF-A thus may have functions in the organisation of chromosomal DNA in addition to its suggested role in hnRNA metabolism.It is widely believed that DNA in eukaryotic interphase nuclei is organized into loop domains with average lengths of 50-100 kb and that these chromatin loops are bound to internal nuclear structures via the scaffold-attachment region (SAR; for review see [l]) or the matrix-associated region (MAR; for review see 12, 31) DNA elements. Many DNA fragments which specifically bind to internal nuclear structures have been identified, but only little is known about their function in vivo and how they exert this function. In addition, only a few proteins ([4, 51 and references therein) are known which possibly are involved in the binding of these DNA fragments to internal nuclear structures.We have therefore attempted to analyse for nuclear proteins which specifically bind SAR DNA elements and we reported recently on the purification of a DNA binding protein with this property 161. This protein, SAF-A (scaffoldattachment factor A), is a component of the nuclear matrix and of scaffolds; it specifically binds vertebrate SAR DNA elements and reconstitutes looped structures with these DNAs [6]. SAF-A is an abundant protein and the large number of molecules presenthucleus indicate a structural function of this protein. In this communication, we identify SAF-A as hnRNP-U [7], a protein component of heterogeneous nuclear ribonucleoprotein complexes (for review see [S-lo]). We refer to this protein as hnRNP-U/SAF-A and characterize the nucleic-acid binding properties of the protein. We further show by ultraviolet (UV) cross-linking experiments that the protein is bound to DNA in vivo and disCorrespondence to A. Richter,
We have compared the influence of spectinomycin (Spc) on individual partial reactions during the elongation phase of translation in vitro by wild‐type and mutant ribosomes. The data show that the antibiotic specifically inhibits the elongation factor G (EF‐G) cycle supported by wild‐type ribosomes. In addition, we have reproduced the in vivo Spc resistant phenotype of relevant ribosome mutants in our in vitro translation system. In particular, three mutants with alterations at position 1192 in 16S rRNA as well as an rpsE mutant with an alteration of protein S5 were analysed. All of these ribosomal mutants confer a degree of Spc resistance for the EF‐G cycle in vitro that is correlated with the degree of growth rate resistance to the antibiotic in culture.
Simian Virus 40 (SV40) infected cells were pulse labeled with (3H) thymidine and chased either in the absence or in the presence of the cytotoxic drug VM26 (teniposide). We investigated the structure of labeled SV40 DNA and found that VM26 had no significant effect on replicative chain elongation but strongly inhibited the conversion of late replication intermediates to mature DNA daughter molecules. The late replicative SV40 DNA intermediates which accumulate in VM26 treated cells contained essentially full length labeled DNA strands. These newly synthesized strands were not part of two catenated interlocked SV40 monomers suggesting that the block occurred prior to the final ligation reaction. Since VM26 is known to be a specific inhibitor of DNA topoisomerase II we conclude that this enzyme is dispensable for the chain elongation of replicating SV40 DNA, but that it is essential for the termination of SV40 DNA replication cycles.
Abstract. RNA polymerase I preparations purified from a rat hepatoma contained DNA topoisomerase activity. The DNA topoisomerase associated with the polymerase had an Mr of 110000, required Mg2+ but not ATP, and was recognized by anti-topoisomerase I antibodies. When added to RNA polymerase I preparations containing topoisomerase activity, anti-topoisomerase I antibodies were able to inhibit the DNA relaxing activity of the preparation as well as RNA synthesis in vitro. RNA polymerase II prepared by analogous procedures did not contain topoisomerase activity and was not recognized by the antibodies. The topoisomerase I: polymerase I complex was reversibly dissociated by column chromatography on Sephacryl S200 in the presence of 0.25 M (NH 4 hS04. Topoisomerase I was immunolocalized in the transcriptionally active ribosomal gene complex containing RNA polymerase I in situ. These data indicate that topoisomerase I and RNA polymerase I are tightly complexed both in vivo and in vitro, and suggest a role for DNA topoisomerase I in the transcription of ribosomal genes.
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