Interactions of a Purified Non‐Histone Chromosomal Protein with DNA and Histone

Shooter, K. V.; Goodwin, Graham H.; Johns, Ernest W.

doi:10.1111/j.1432-1033.1974.tb03690.x

Cited by 96 publications

(57 citation statements)

References 14 publications

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“…Given these results, we reasoned that good candidates for the source of the activity in the HeLa extracts are a subset of the HMG proteins. HMG1 and HMG2, like HU, are relatively abundant, bind D N A without sequence specificity, and resist heat and acid treatment (Shooter et al 1974;Goodwin et al 1975;Johns 1982;Kuehl et al 1984;Drlica and Rouvihre-Yaniv 1987). HMG1 and HMG2 are -2 5 kD in molecular mass but migrate at -2 9 kD on SDS-polyacrylamide gels.…”

Section: Hmg Proteins In Hela Nuclear Extract Can Substitute For H U mentioning

confidence: 99%

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

1993

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The mammalian high mobility group proteins HMG1 and HMG2 are abundant, chromatin-associated proteins whose cellular function is not known. In this study we show that these proteins can substitute for the prokaryotic DNA-bending protein HU in promoting the assembly of the Hin invertasome, an intermediate structure in Hin-mediated site-specific DNA inversion. Formation of this complex requires the assembly of the Hin recombinase, the Fis protein, and three cis-acting DNA sites, necessitating the looping of intervening DNA segments. Invertasome assembly is strongly stimulated by HU or HMG proteins when one of these segments is shorter than 104 bp. By use of ligase-mediated circularization assays, we demonstrate that HMG1 and HMG2 can bend DNA extremely efficiently, forming circles as small as 66 bp, and even 59-bp circles at high HMG protein concentrations. In both invertasome assembly and circularization assays, substrates active in the presence of HMG1 contain one less helical turn of DNA compared with substrates active in the presence of HU protein. Analysis of different domains of HMG1 generated by partial proteolytic digestion indicate that DNA-binding domain B is sufficient for both bending and invertasome assembly. We suggest that an important biological function of HMG1 and HMG2 is to facilitate cooperative interactions between cis-acting proteins by promoting DNA flexibility. A general role for HMG1 and HMG2 in chromatin structure is also suggested by their ability to wrap DNA duplexes into highly compact forms.

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Section: Hmg Proteins In Hela Nuclear Extract Can Substitute For H U mentioning

confidence: 99%

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

1993

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“…[9] of '"1-labelled protein HMGZ with the unlabelled protein HMGZ Preparation of DNA DNA was isolated from bacteriophage T7 by the method described by Lawley et al [3]. Calf thymus DNA was prepared by the method of Kay et al [4].…”

Section: Preparation Of Non-histone Protein Hmg2mentioning

confidence: 99%

Interaction of a Non‐Histone Chromatin Protein (High‐Mobility Group Protein 2) with DNA

Goodwin

Shooter

Johns

1975

European Journal of Biochemistry

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1. The interaction with DNA of the calf thymus chromatin non-histone protein termed the highmobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 1251-labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263-2701 for the interaction of highmobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high-mobility group protein 2 differs from high-mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form.2. The molecular weight of high-mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26000 was obtained.3. A low level of nuclease activity detected in one preparation of high-mobility group protein 2 has been investigated.In a previous paper [I] the properties of one of a group of non-histone proteins from calf thymus nucleoprotein, high-mobility group protein 1 (protein HMGl), have been described and the characteristics of its interaction with DNA investigated using analytical sedimentation techniques. It was found that the binding to nucleic acid was reversible, the addition of more DNA to a mixture leading to requilibration of all bound protein molecules among all DNA molecules within the time scale of the measuring period. Using a simple theory to relate the observed increase in sedimentation rate of the DNA to the amount of protein bound, estimates of the equilibrium constant for the binding reaction and of the size of the binding site were made. These results suggested that there was little difference in the binding parameters of the protein to homologous or heterologous DNA but that the protein was bound less strongly to denatured DNA. Similar studies have now been made of the interaction of nucleic acids and a second non-histone protein from calf thymus, high-mobility group protein 2 (protein HMG2). Preliminary measurements on the binding to bacteriophage T7 DNA showed that, with this protein, the Abbreviation. HMG, high-mobility group.Eur. J. Biochem. 54 (1975) increase in sedimentation rate was greater than could be accounted for simply in terms of the amount of protein bound, i.e. some change in the overall shape of the DNA molecule was also involved. These observations made it necessary to use an alternative method of measuring the amounts of free and bound protein and for this purpose a sample of one preparation of the protein was labelled with lz5I.The earlier work on the interaction of protein HMGI with calf thymus DNA had suggested that the binding might be dependent on the molecular weight of the DNA. To test this possibility the interaction of protein HMG2 with bacteriophage T7 DNA degraded by ultrasonic irradiation has been investigated. MATERIALS AND METHODS Preparation of Non-Histone Protein HMG2Calf thymus chromatin non-histone proteins were fractionated by trichloroacetic acid precipitation, and the HMG proteins separ...

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“…They possess 40 -50 % a-helix structures which are sensitive to pH as well as urea concentration [3,4]. Shooter et al [2] and Goodwin et al [5] showed that proteins HMGl and HMG2 bound DNA in a similar manner through ionic bonding between the basic amino acid Abbreviation. CD, circular dichroism ; proteins HMG 1 and HMG2, high-mobility-group non-histone proteins 1 and 2. residues and the phosphates of the DNA.…”

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Interaction of Non-histone Chromosomal Proteins HMG1 and HMG2 with DNA

Goodwin

et al. 1977

Eur J Biochem

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Interaction between non-histone protein HMGl or HMG2 and DNA has been studied by using thermal denaturation and circular dichroism (CD) spectroscopy. We have made the following observations.1. The binding of each of these two proteins to DNA stabilizes the latter, as shown by an increase in melting temperature of 20 "C (from 45 "C to about 65 "C).2. There are 6.0 amino acids/nucleotide in HMGl-bound DNA and 5.0 in HMGZbound DNA which suggests that each HMGl molecule would cover about 20 base pairs of DNA and each HMG2 molecule would cover about 25 base pairs.3. The a-helical content of these two non-histone proteins in the complexes, estimated from the CD value at 220 nm, is about one third to one half that of total proteins in calf thymus chromatin.4. DNA conformation is distorted only slightly by the binding of protein HMGl or HMG2. 5.Neither the melting nor the CD properties of HMGl . DNA or HMG2 . DNA complexes differ substantially whether they are prepared by NaC1-gradient dialysis in urea or by direct mixing of protein and DNA at 0.15 M NaC1, followed by dialysis against the same buffer i.e. 0.25 mM EDTA (pH 8.0).Most of the physical studies of chromatin have so far emphasized the interaction between histones and DNA. However, it is evident that the non-histone chromosomal proteins also play an important part in the structure and function of chromatin. Since these proteins are generally quite different from the histones (e. g. having lower isoelectric points) the interaction of non-histone proteins with DNA could be quite different. In view of the importance of possible non-histone protein-DNA interactions in chromatin this study was initiated to investigate the interaction with DNA of two purified DNA-binding nonhistone proteins, proteins H M G l and HMG2 from calf thymus chromatin. These two proteins are extracted from chromatin with 0.35 M NaCl, contain 24-26 % lysine and arginine residues and 29-31 % aspartic and glutamic residues [l, 21. They possess 40 -50 % a-helix structures which are sensitive to pH as well as urea concentration [3,4]. Shooter et al.[2] and Goodwin et al. [5] showed that proteins HMGl and HMG2 bound DNA in a similar manner through ionic bonding between the basic amino acid Abbreviation. CD, circular dichroism ; proteins HMG 1 and HMG2, high-mobility-group non-histone proteins 1 and 2.residues and the phosphates of the DNA. Although there is no direct evidence for the association of HMG proteins with DNA in chromatin, the high content of basic amino acids as in the histones suggests that they are likely to be nucleic-acid-bound. They do not appear to be bound to RNA since extensive digestion of chromatin with ribonuclease does not release HMG proteins from chromatin [6] (and our own unpublished observations).Interaction of non-histone proteins, HMG 1 and HMG2, with DNA has been studied using thermal denaturation and circular dichroism (CD) spectroscopy. The thermal denaturation method has been used successfully in the studies of structural properties of chromatin, such as thermal s...

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Interactions of a Purified Non‐Histone Chromosomal Protein with DNA and Histone

Cited by 96 publications

References 14 publications

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

Interaction of a Non‐Histone Chromatin Protein (High‐Mobility Group Protein 2) with DNA

Interaction of Non-histone Chromosomal Proteins HMG1 and HMG2 with DNA

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