No abstract

Two chromatin nonhistone proteins (from calf thymus) of the high mobility group, HMG1 and HMG2, reduce the linking number (topological winding number) of a circular DNA if the covalent closure of the DNA is carried out in their presence. This indicates that these proteins can either unwind the double helix, or induce a supercoiling of the DNA.

The distribution of DNA topoisomerase I within Drosophila polytene chromosomes was observed by immunofluorescent staining with affinity-purified antibodies. The enzyme is preferentially associated with active loci, as shown by prominent staining of puffs. The heat shock loci 87A-87C are stained after, but not before, heat shock induction. A detailed comparison of the distribution of topoisomerase I with that of RNA polymerase II reveals a similar, al, though not identical, pattern of association. Topoisomerase I is also found in association with the nucleolus, the site of transcription by RNA polymerase I.The polytene chromosomes of the Drosophila salivary gland provide an excellent system for investigating the distribution of specific chromosomal proteins. Although highly organized, these giant chromosomes behave like diploid interphase chromatin in many assays of function and fine structure (1-6). They can, however, be easily observed under the light microscope; some substructure, such as transcriptionally active sites, can be recognized. It is possible to localize proteins in these chromosomes and thereby obtain some information as to the biological processes in which the given proteins might be involved. In order to visualize the chromosomal proteins, the method of indirect immunofluorescence was developed several years ago (7,8). The distribution patterns of a number of chromosomal proteins of unknown function have been determined by this approach (8-11). The method has allowed identification of a subclass of nonhistone chromosomal proteins that are prominently associated with loci that are active or inducible at some time in the salivary glands of the third instar larvae and prepupae (10,12,13).In addition, distribution patterns of proteins of known function have been analyzed-e.g., RNA polymerase 11 (14-17) and ribonucleoproteins (18). These proteins are preferentially associated with the transcriptionally active regions of the genome. In this paper we report the distribution pattern of DNA topoisomerase I. This enzyme is well characterized at the molecular level, but the range of its biological functions in eukaryotes has yet to be established (for reviews, see refs. 19-25). The ability of eukaryotic topoisomerase I to relax either negatively or positively supercoiled DNA hints that the enzyme might play a role in gene activation, either effecting structural changes in chromatin as it assumes a more "open" conformation (e.g., as detected by the appearance of puffs) and/or facilitating transcription per se (26, 27). There is some evidence in favor of a role of topoisomerase in transcription. For example, topoisomerase has been shown to be associated with ribosomal gene chromatin actively expressing rRNA (28); topoisomerase I is recovered with nucleosomes in a fraction enriched for transcriptionally active genes (29). Complex formation between eukaryotic DNA topoisomerase I and chromosomal high mobility group proteins and histone H1 has also been reported (30). However, there has been no direct evi...

The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobilitygroup protein 1 with DNA has also been studied. 1.Circular dichroism results indicate that in the presence of salt both proteins are 40-50 % helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4.2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding.3. Nuclear magnetic resonance spectra of complexes between high mobility group protein 1 and DNA demonstrate that at low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA.Chromatin contains a group of non-histone proteins which are characterised by their high content of acidic and basic amino acids [l]. Several of these proteins, which we have termed the high-mobilitygroup (HMG) proteins have been isolated in a pure form from calf thymus chromatin [1,2]. Of particular interest are the two most highly charged proteins, high-mobility-group 1 and high-mobility-group 2, which contain 25 % basic amino acids and 30 % acidic amino acids. Judging by this unusual feature and that there are about lo6 molecules of each per cell nucleus, it is probable that, like the histones, these are chromatin structural proteins binding to the DNA by means of ionic bonds between the basic amino acids and the phosphate groups of DNA [3,4].The utilisation of several spectroscopic techniques such as infrared, circular dichroism (CD) and most particularly high-resolution nuclear magnetic resonance (NMR) has enhanced our understanding of the solution conformations of histones and protamines Abbreviations. NMR, nuclear magnetic resonance; CD, circular dichroism. both in the presence and absence of DNA [5]. Of the five main histone fractions, all but the most lysinerich (Hl) are observed to take up a partially helical structure on salt addition at pH 3-4 with the simultaneous formation of inter-histone aggregates that include most of the molecule but leave the most basic terminal regions free in the solution. The aggregating capacity of the individual histones is probably related to a functional self-assembly property and recent results on associations between histones of different type strongly support the concept of histone selfassembly [6]. An increase in the pH from 3-7 does not by itself induce large structural changes in the conformation of these four histones but this is not found to be the case with H1 which is observed to form both secondary and tertiary structure on pH increase without any concomitant aggregation [7]. At neutral pH (or at pH 3 in high salt) H1 therefore behaves like a single subunit globular protein with the notable difference that only about half of...

In a previous communication we have shown that both HMG1 and HMG2 nonhistone proteins change the DNA helical structure and the binding of HMG1 and HMG2 to DNA induces a net unwinding equivalent of DNA double helix (Javaherian, K., Liu, L. F. and Wang, J. C. (1978) Science, 199, 1345-1346). Employing melting absorption technique, we now show that in the presence of salt HMG1 and HMG2 destabilize DNA whereas in the absence of salt, they both stabilize DNA molecules. Consequently the folded structure of HMG must play an important role in melting DNA. Furthermore, by measuring topological winding number using competition unwinding experiments, we conclude that HMG1 has a higher affinity for a single-stranded DNA relative to double-stranded DNA. These results together suggest that HMG1 and HMG2 unwind DNA double helix by local denaturation of the DNA base pairs. The net unwinding angles have been measured to be 22 degrees and 26 degrees per molecule of HMG1 and HMG2 respectively.