Phosphorylation alters the affinity of high mobility group protein HMG 14 for single-stranded DNA

The effect of phosphorylation on the affinity of HMG 14 from calf thymus for single-stranded DNA (ssDNA) was studied, using a cyclic GMP-dependent protein kinase from bovine lung and a nuclear protein kinase II from rat liver. When phosphorylated by G-kinase, HMG 14 eluted at 0.27 M NaCl from the ssDNA-column, whereas the native protein eluted at 0.30 M salt concentration. In contrast, phosphorylation by nuclear protein kinase II did not alter dissociation of HMG 14 from ssDNA and the phosphoprotein consequently coeluted with the native HMG 14. Thus, addition of a negative charge by phosphorylation of the Ser-6 residue by G-kinase presumably weakens the interaction between the DNA-binding amino acids of HMG 14 and the negatively charged phosphate groups of DNA.     

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.     

Nonhistone chromosomal protein HMG 1 interactions with DNA. Fluorescence and thermal denaturation studies

The interaction of high mobility group protein 1 (HMG 1) isolated from chicken erythrocytes with DNA has been characterized using the intrinsic tryptophan fluorescence of the protein as a probe. It was found that the fluorescence is quenched approximately 30% upon binding to either single- or double-stranded DNA. Fluorescent titrations indicate that the physical site size for HMG 1 binding on native DNA is approximately 14 base pairs (or 14 bases for binding to single-stranded DNA). Binding to single-stranded poly(dA) is only slightly dependent on ionic strength, although the affinity for double-stranded DNA is strongly ionic strength-dependent and has an optimum at approximately 100-120 mM Na+. Above this range, binding to native DNA is virtually all electrostatic in nature. Although the affinity of HMG 1 for single-stranded DNA is higher than that for double-stranded DNA at the extremes of the ionic range studied, no clear evidence for a helix-destabilizing activity was obtained. At low ionic strength, the protein actually stabilized DNA against thermal denaturation, while at high ionic strength, HMG 1 appears to undergo denaturation below the Tm of the DNA. Studies of the environment of the tryptophan fluorophores using collisional quenchers iodide, cesium, and acrylamide suggest that the predominant fluorophore is relatively exposed but constrained in a rigid, positively charged environment.     

HMG17 protein facilitates the DNA catenation reaction catalyzed by DNA topoisomerases

HMG17 protein is shown to greatly facilitate the catention of double-stranded DNA rings catalyzed by DNA topoisomerases. Even at low DNA concentrations such that catenanes are not observable in the absence of HMG17, the addition of the protein promotes the catenation of greater than 95% of the input DNA into networks that do not enter the gel upon electrophoresis. Electron microscopy and restriction enzyme cleavage experiments indicate that these networks are large structures containing many catenated DNA rings. The HMG17-promoted DNA network formation has been observed with calf thymus type II DNA topoisomerase and the type I topoisomerases of Escherichia coli, Micrococcus luteus, and calf thymus.     

Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation

A rat was irradiated to the upper abdomen including the liver and then partially hepatectomized. The subsequent synthesis and phosphorylation of histone H1 and nonhistone chromosomal high mobility group (HMG) proteins were investigated. Incorporation of [3H]lysine into histone H1 was increased and reached its peak at 27 h after hepatectomy, and 14 Gy of X rays inhibited the increase. Increase in the incorporation of [3H]lysine into HMG (1 + 2), 14, and 17 which occurred around 27 h after hepatectomy was not inhibited by 14 Gy irradiation. Phosphorylation of histone H1, measured with 32Pi incorporation in vivo, was maximal between 21 and 24 h, and it was inhibited by 4.8 Gy of X rays and delayed with 1.9 Gy. Phosphorylation of HMG 14, which was the only HMG protein phosphorylated under present conditions, was not affected by X irradiation. The [3H]thymidine incorporation into nuclear DNA started increasing at 21 h and reached its maximum at 27 h after hepatectomy. X irradiation with 4.8 Gy inhibited the incorporation, and 1.9 Gy lowered it.     

Phosphorylation of high-mobility-group chromatin proteins by protein kinase C from rat brain

Chromosomal high-mobility-group (HMG) proteins have been examined as substrates for calcium/phospholipid-dependent protein kinase C. Protein kinase C from rat brain phosphorylated efficiently both HMG 14 and HMG 17 derived from calf thymus and the reactions were calcium/phospholipid-dependent. About 1 mol of ³²P was incorporated per mol of HMG 14 and HMG 17. Phosphopeptide mapping suggested that the same major site was phosphorylated in both proteins at serine. The apparent K_m values for HMG 14 and HMG 17 were about 5 μM. HMG 14, HMG 17 and the five histone H1 subtypes prepared from rat thymus, liver and spleen were phosphorylated by the kinase. HMG 14 and HMG 17 from transformed human lymphoblasts (Wi-L2) were also phosphorylated in a calcium/phospholipid-dependent manner. HMG 1 and HMG 2 from the tissues examined were found to be poor substrates for the kinase.     

Preferential affinity of high molecular weight high mobility group non-histone chromatin proteins for single-stranded DNA.

We have subjected proteins dissociated from chicken erythrocyte or calf thymus chromatin by 0.35 M NaCl to sequential chromatography on columns containing immobilized double-stranded DNA and single-stranded DNA. At 0.2 M NaCl, 1 mM Tris . Cl (pH 7.5), the high molecular weight, high mobility group proteins (HMG-1, HMG-2, and HMG-E), were not retained by double-stranded DNA columns, but were retained by single-stranded DNA columns. Thus, in that solvent, those proteins exhibit selective affinity for single-stranded DNA. This suggests that the functions of the high molecular weight, high mobility group proteins might involve destabilizing the DNA double helix by virtue of their preferential affinity for single-stranded DNA.     

Thermal denaturation and fluorescence study of nucleosomes containing non-histone chromosomal protein HMG2

Interaction of calf thymus non-histone chromosomal protein HMG2 with H1,H5-depleted nucleosomes from chicken erythrocytes was studied by means of thermal denaturation and an N-(3-pyrene)maleimide fluorescence probe. Under low ionic conditions (2 mM Tris buffer plus EDTA) addition of 1-2 molecules of HMG2 per nucleosome markedly stabilized the segment of the linker DNA against thermal denaturation. Under approximately physiological ionic conditions (0.1 M NaCl) addition of two HMG2 molecules per nucleosome, labeled by N-(3-pyrene)maleimide at the sulfhydryl groups of Cys-110 of histones H3, resulted in a decrease of the pyrene excimer fluorescence corresponding to the slight movement of the sulfhydryl groups of the two histone H3 molecules apart.     

Polyamines and heparin do not appreciably influence phosphorylation of chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II

Phosphorylation of acidic substrates such as casein and phosvitin by nuclear protein kinase II is stimulated by polyamines and inhibited by heparin, which mimics an endogenous proteoglycan inhibitor. The phosphorylation in vitro of the chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II were examined in this study focusing on the modifying effects of polyamines and heparin. Both HMG proteins were phosphorylated by the enzyme, but polyamines did not appreciably influence the extent of their phosphorylation. In addition, heparin did not inhibit the kinase reaction with the HMG proteins as substrates. These results indicate that the nuclear protein kinase II does actively phosphorylate HMG 14 and HMG 17 in vitro but that in contrast to some model substrates, polyamines and heparin do not appreciably affect their phosphorylation.     

The role of H1 histone phosphorylation in the cell cycle : Turbidity studies of H1-DNA interaction

A semi-automatic turbidimetric method was used to study the interaction of H1 histone with DNA. Phosphorylation of H1 by a growth-associated kinase had two effects on the interaction. At high salt (0.4 M to 0.6 M NaCl) phosphorylated H1 is released from DNA at lower salt concentration than control H1, but at moderate salt (0.1–0.3 M NaCl) phosphorylated H1 cross-links DNA more effectively (higher turbidity) than unphosphorylated H1. The second effect was not observed with H1 phosphorylated at two other sites and the results are interpreted as providing support for the previous proposal that growth-associated H1 phosphorylation initiates chromosome condensation in prophase of the cell cycle.     

Nonhistone chromatin proteins HMG-14 and HMG-17 bind preferentially to single-stranded DNA.

Proteins extracted from chicken erythrocyte chromatin with 0.35 M NaCl were subjected to sequential chromatography on columns containing immobilized double-stranded and single-stranded DNA's. Two-dimensional electrophoresis of protein fractions revealed that HMG-14 and HMG-17 are among the proteins that are retained by the single-stranded DNA column in 0.2 M NaCl/l mM Tris-Cl (pH 7.5) after having failed to be retained by the double-stranded column under the same conditions. That suggests that those two proteins possess preferential affinity for single-stranded DNA. Further evidence for that was provided by chromatography of purified HMG-14 and of purified HMG-17 on single-stranded and double-stranded DNA columns. We discuss the possible relevance of our results to suggested functions of HMG-14 and HMG-17.     

Binding of HMG14 non-histone protein to histones H2A, H2B, H1 and DNA in reconstituted chromatin

The interaction between calf thymus HMG14 and rat liver chromatin components has been studied via reconstitution and chemical cross-linking. Selective labeling of HMG14 with photoactivable reversible heterobifunctional reagents has allowed a clear identification of the histones interacting with it (histones H2A, H2B and H1). These results are not dependent on whether the chromatin samples used were bulk chromatin, mononucleosomes, or core particles (for H2A and H2B). In addition to histone proteins, DNA also seems to be involved in HMG14 attachment to nucleosome.     

High mobility group proteins in ram spermatids

The four major high mobility group proteins HMG 1, 2, 14 and 17, HMG 19B and histone H1(0) were identified in the ram testis by their extraction and solubility characteristics and by their electrophoretic mobilities. HMG 14 and 17 were isolated by chromatography and amino acid analysis revealed that they were similar to their calf thymus analogues. A protein, named 2R and co-extracted with HMG 14, was also purified and analysed. Electrophoretic analyses of the proteins extracted by 0.75 M perchloric acid (PCA) or by 0.35 M NaCl from round and non-round spermatids, separated by centrifugal elutriation, showed that the four major HMG proteins disappear from nuclei in the oldest round spermatids, at the time the nuclear content of protein 2R and histone H1(0) increases in spermatids. Ubiquitin and HMG 19B were present in the round and elongating spermatids, but not in elongated spermatids which contained only protamine. The relation was considered between several protein changes and genetic inactivation and structural reorganization of the spermatid chromatin.     

The HMG1 ta(i)le

We have studied structural changes in DNA/protein complexes using the CD spectroscopy, upon the interaction of HMG1-domains with calf thymus DNA at different ionic strengths. HMG1 protein isolated from calf thymus and recombinant HMG1-(A+B) protein were used. Recombinant protein HMG1-(A+B) represents a rat HMG1 lacking C-terminal acidic tail. At low ionic strength (15 mM NaCl) we observed similar behavior of both proteins upon interaction with DNA. Despite this, at higher ionic strength (150 mM NaCl) their interaction with DNA leads to a completely different structure of the complexes. In the case of HMG1-(A+B)/DNA complexes we observed the appearance of DNA fractions possessing very high optical activity. This could be a result of formation of the highly-ordered DNA structures modulated by the interaction with HMG1-domains. Thus the comparison studies of HMG1 and HMG1-(A+B) interaction with DNA show that negatively charged C-terminal tail of HMG1 modulates interaction of the protein with DNA. The striking difference of the behaviour of these two systems allows us to explain the functional role of multiple HMG1 domains in some regulatory and architectural proteins.     

Amino acid sequence of histone H1 at the ADP-ribose-accepting site and ADP-ribose X histone-H1 adduct as an inhibitor of cyclic-AMP-dependent phosphorylation

The ADP-ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP-ribosyltransferase was determined and effects of the ADP-ribose X histone-H1 adduct on cAMP-dependent phosphorylation of the histone H1 were investigated. ADP-ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate-32P]NAD and the purified ADP-ribosyltransferase. N-Bromosuccinimide-directed bisection of ADP-ribosylated histone H1 showed that the NH2-terminal fragment (Mr = 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP-dependent protein kinase. Digestion of the NH2-terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high-performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29-34 of histone H1, containing the arginine residue as the ADP-ribosylation site. These results indicate that ADP-ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2-terminal side of the phosphate-accepting serine residue 38. Phosphorylation of histone H1 from calf thymus by cAMP-dependent protein kinase was markedly reduced when histone H1 was ADP-ribosylated. Kinetic studies of phosphorylation revealed that ADP-ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non-competitive inhibitor of ATP.     

Unwinding of DNA by nonhistone chromosomal protein HMG(1 + 2) from pig thymus as determined with endonuclease

A high mobility group (HMG) nonhistone protein fraction HMG(1 + 2), composed of HMG1 and HMG2, was prepared from pig thymus chromatin. In order to examine a possibility that the HMG(1 + 2) participates in the unwinding of the DNA double-helix, DNA hydrolysis assay systems with the endonucleases specific for single-stranded DNA were employed. In the presence of HMG(1 + 2), the hydrolysis of double-stranded DNA by N. crassa endonuclease was markedly promoted, while the hydrolysis of single-stranded DNA was hardly enhanced. The reaction kinetic data showed that the stimulation of the hydrolysis of double-stranded DNA in the presence of HMG(1 + 2) was due to the unwinding of the DNA double-helix by the HMG(1 + 2), and not due to stimulation of enzyme activity of the endonuclease by the protein. The unwinding reactions were dependent on the HMG protein concentration at low weight protein to DNA ratios and reached a maximum at the ratio of 0.025. The region unwound in the whole DNA was partial. Similar results were obtained for experiments with nuclease S1. Isolated HMG1 and HMG2 fractions showed DNA unwinding activity of similar extents. The association constant obtained by fluorescence quenching analysis showed that the HMG(1 + 2) has higher affinity to single-stranded DNA than to double-stranded DNA. The susceptibility to the unwinding differed with the DNA source. These results suggest that HMG(1 + 2) at a low weight protein to DNA ratio binds to some limited double-stranded region in DNA and unwinds the DNA partially.     

The in vitro reconstitution of nucleosome and its binding patterns with HMG1／2 and HMG14／17 proteins

Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociatedfrom DNA at 1M NaC1. When the salt concentration was slowly reduced to 650 mM and 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic “beads-on-a-string“ structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution of nucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5‘flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.