Structure of eukaryotic DNA binding sites for steroid hormone-apolipoprotein A-I complexes

High affinity for DNA and synthetic oligonucleotides was detected for apolipoprotein A-I (ApoA-I) by affinity chromatography, affinity modification, and enzymatic analysis. Competitive inhibition and Southern hybridization showed that the tetrahydrocortisol (THC)-ApoA-I complex specifically bound to high-molecular-weight DNA in regions containing GCC/CGG sequences. The CC(GCC)₃ · GG(CGG)₃ duplex was found to be sensitive to nuclease S1 under the action of the THC-ApoA-I complex. The eukaryotic DNA binding sites for steroid (THC, androsterone)-ApoA-I complexes were found to be involved in the initiation of DNA copying in vitro.     

Characteristics of the interactions of cortisol-apolipoprotein A-I and tetrahydrocortisol-apolipoprotein A-I complexes with eukaryotic DNA

Primary hepatocyte culture has been used to demonstrate that the cortisol-apolipoprotein A-I complex does not affect the DNA and protein biosynthesis rates, whereas the tetrahydrocortisol-apolipoprotein A-I complex (THC-apoA-I) substantially increases the rates of ³H-thymidine and ¹⁴C-leucine incorporation into DNA and protein, respectively. Small-angle X-ray scattering data show that only THC-apoA-I effectively interacts with eukaryotic DNA, which is accompanied by local DNA melting. The (GCC)_n repeat, a component of many human and other eukaryotic genes, is the most probable region of the interaction of this complex with DNA. An oligonucleotide (duplex) of this type has been synthesized. Its interaction with THC-apoA-I yields a larger complex, which breaks up, giving rise to complementary oligonucleotide strands. They also interact with THC-apoA-I. The equilibrium kinetics of this multiphase process is described. The interaction of the cortisol-apolipoprotein A-I complex with the duplex is less specific and does not cause its breakup or the formation of complementary oligonucleotides.     

Features of interaction of complexes cortisol-apolipoprotein A-I and tetrahydrocortisol-apolipoprotein A-I with eukariotic DNA

On primary culture of hepatocytes it is shown, that a complex cortisol-apolipoprotein A-I did not change rate of biosynthesis DNA and protein, whereas the complex tetrahydrocortisol-apolipoprotein A-I (THC-apoA-I) essentially raised rate of incorporation 3H-thymidine in DNA and 14C-leucine into protein. By a method of small-angle X-ray scattering it is shown, that appreciable interaction with eukariotic DNA is marked only in case of use of a complex THC-apoA-I, thus there is local fusion of DNA. The most probable region of interaction of the given complex with DNA is repetition (GCC)n the type, included in structure of many genes eukariot, including the human. It is synthesized oligonucleotid (duplex) of this type. It is shown, that at his interaction with complex THC-apoA-I there is a formation of more difficult complex, which breaks up with formation of complementary chains of oligonucleotides. The last also enter interaction with complex THC-apoA-I. It is given of kinetic this multiphasic process. Interaction of a complex cortisol-anoA-I with a duplex is less specific and does not result reduce in decay of the duplex and in formation of complementary oligonucleotides.     

Tetrahydrocortisol-apolipoprotein A-I complex specifically interacts with eukaryotic DNA and GCC elements of genes

Tetrahydrocortisol stimulates DNA and protein biosynthesis in hepatocytes only when it enters the complex with apolipoprotein A-I. Tetrahydrocortisol–apolipoprotein A-I (THC–apoA-I) complex specifically interacts with eukaryotic DNA isolated from rat liver. In the process of interaction, rupture of hydrogen bonds between the pairs of nitrous bases occurs with the formation of single-stranded DNA structures. In such state DNA forms complexes with DNA-dependent RNA-polymerase. The most probable site of binding the tetrahydrocortisol–apolipoprotein A-I complex with DNA is the sequence of CC(GCC)_n type entering the structure of many genes, among them the structure of human apolipoprotein A-I gene. Oligonucleotide of this type has been synthesized. Association constant (K_ass) of it with tetrahydrocortisol–apolipoprotein A-I complex was shown to be 1.66×10⁶ M⁻¹. Substitution of tetrahydrocortisol for cortisol in the complex results in a considerable decrease of K_ass. It was assumed that in the GC-pairs of the given sequence tetrahydrocortisol itself participates in the formation of hydrogen bonds with cytosine, favoring their rupture with complementary base—guanine.     

Interaction of tetrahydrocortisol-apolipoprotein A-I complex with eukaryotic DNA and with single-stranded oligonucleotides

The complex formed by tetrahydrocortisol (THC) and apolipoprotein A-I (ApoAI) specifically interacts with eukaryotic DNA from rat liver. Taken together, physical and chemical data and the results of small-angle X-ray scattering analysis show that interaction of the THC-ApoAI complex with eukaryotic DNA results in deformation of the DNA double helix. Single-stranded fragments were demonstrated to cause deformation of the double helix. In this state DNA forms complexes with DNA-dependent RNA polymerase. This interaction is cooperative and of saturating type; up to six enzyme molecules bind with one DNA molecule. The putative site of complex binding with DNA is the sequence CC(GCC)n found in many genes including the human ApoAI gene. An oligonucleotide of this type was synthesized. Its association constant (Ka) was 1.66 x 10(6) M-1. Substitution of THC with cortysol considerably decreases the Ka. We suggest that THC interacting with GC pairs of the binding site forms hydrogen bonds with cytosine, inducing rupture of the bonds within the complementary nucleic base pair.     

Mechanisms of interaction of tetrahydrocortisol and its complex with apolipoprotein A-I and DNA regulatory cis-elements of CC(GCC)5/GG(CGG)5 type

An IR-spectroscopy study of the mechanism of interaction between duplex CC(GCC)5/GG (CGG)5Li2 and tetrahydrocortisol or tetrahydrocortisol-apolipoprotein A-I complex revealed the formation of hydrogen bonds between the OH group of the tetrahydrocortisol A-ring and the C=0 group of cytosine or guanine. Tetrahydrocortisol forms hydrogen bonds with the PO2-group of the duplex and with the OH-group of monosaccharide. The interaction of tetrahydrocortisol and apolipoprotein A-I with the duplex occurs at the same active site, namely, with the C=O-group of bases. The order --> order structural transition takes place in the duplex under the action of tetrahydrocortisol. The order --> disorder structural transition takes place in the duplex under the action of tetrahydrocortisol-apolipoprotein A-I complex.     

鉴定水稻中一个新的专一结合GCC元件的AP2/EREBP族转录因子

TSH1,是通过搜寻GenBank的EST库而获得的一个来源于水稻的含AP2/EREBP保守结构域的蛋白质.为了详细分析TSH1蛋白与其DNA顺式元件的结合特性,首先采用传统的凝胶阻滞实验和酵母单杂交技术,证实TSH1在体内和体外均专一性地结合于GCC元件,然后利用原子力显微镜技术精确测量了TSH1蛋白与GCC元件在单分子水平的相互作用力.结果表明,GST-TSH1与DRE元件没有特异性的结合,而GST-TSH1与GCC元件结合力的大小为(83.9±2.2)pN,这种特异性的结合可以被加入的游离TSH1蛋白明显降低.GST蛋白和突变GCC元件作为负对照显示出与GCC元件无特异性作用力.以上结果充分证明,TSH1是专一性地与GCC元件相作用的转录因子,而且原子力显微镜对于检测转录因子与DNA相互作用时单碱基的突变十分灵敏.通过比较几种评估转录因子与DNA顺式元件结合特异性的方法,阐述了原子力显微镜技术的特点及优越性.     

Interaction of Tetrahydrocortisol–Apolipoprotein A-I Complex with Eukaryotic DNA and with Single-Stranded Oligonucleotides

The complex formed by tetrahydrocortisol (THC) and apolipoprotein A-I (ApoAI) specifically interacts with eukaryotic DNA from rat liver. Taken together, physical and chemical data and the results of small-angle X-ray scattering analysis show that interaction of the THC–ApoAI complex with eukaryotic DNA results in deformation of the DNA double helix. Single-stranded fragments were demonstrated to cause deformation of the double helix. In this state DNA forms complexes with DNA-dependent RNA polymerase. This interaction is cooperative and of saturating type; up to six enzyme molecules bind with one DNA molecule. The putative site of complex binding with DNA is the sequence CC(GCC)_n found in many genes including the human ApoAI gene. An oligonucleotide of this type was synthesized. Its association constant (K _a) was 1.66·10⁶ M^–1. Substitution of THC with cortisol considerably decreases the K _a. We suggest that THC interacting with GC pairs of the binding site forms hydrogen bonds with cytosine, inducing rupture of the bonds within the complementary nucleic base pair.     

Effect of tetrahydrocortisol-apolipoprotein A-I complex on RNA polymerase interaction with eukaryotic DNA and the rate of protein biosynthesis in hepatocytes

A mechanism of activation of protein biosynthesis in hepatocytes was proposed as effected by the conditioned medium of nonparenchymal liver cells incubated in the presence of high density lypoproteins, cortisol, and lypopolysaccharides. It was found that the increase in the biosynthesis rate was associated with the formation of the tetrahydrocortisol-apolipoprotein A-I (THC-apoA-I) complex in macrophages, which display 5 alpha- and 5 beta-reductase activity and are constituents of nonparenchymal liver hepatocytes. Using the small-angle X-ray scattering technique, it was shown that the THC-apoA-I-eukaryotic DNA interaction may break hydrogen bonds between pairs of complementary nucleic bases and cause the formation of single-stranded DNA fragments capable of binding to DNA-dependent RNA polymerase. The interaction is highly cooperative and has a saturating mode, up to six enzyme molecules being bound per DNA molecule.     

Interaction of human apolipoprotein A-I with dipalmitoylphosphatidylcholine in vesicular and micellar complexes

The interactions of human apolipoprotein A-I (apo A-I) with dipalmitoylphosphatidylcholine (DPPC) in vesicular complexes at low protein concentrations and in micellar complexes at high protein concentrations are compared. The C-terminal segment of this protein, with a relative molecular weight (Mr) of about 11,000, is protected on trypsin treatment of apo A-I-vesicle complexes. A segment within the sequence from Leu-189 to Arg-215 of apo A-I penetrates the hydrophobic interior of the membrane, as found in a hydrophobic labeling experiment involving 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID). No appreciable stretch of apo A-I in micellar complexes was found to be protected from the tryptic digestion. This indicates that the interactions of apo A-I with lipids in the vesicular and micellar complexes are different. The binding equilibrium of apo A-I as to DPPC vesicles at low protein concentrations, as studied by hydrophobic labeling of the bilayer-penetrating segment, is reached within about 1 h, while the formation of micellar complexes at high protein concentrations takes about 24 h at 42 degrees C. Time-dependent labeling studies involving photoreactive phosphatidylcholine (PC) with high apo A-I concentrations suggested an initial interaction with the head group region of the bilayer followed by interaction with the tail ends of the acyl chains of the lipid. A possible mechanism for the micellization process is discussed.     

Novel DNA-binding ligands with sequence selectivity based on hydrophobic structure.

We have developed diamino-bistetrahydrofuran compounds (diamino-bisTHF) as new DNA binding molecules. Diamino-bisTHF (3:RR8) stabilized GC-rich duplex DNA with sequence specificity. DNA binding affinity increased as the alkyl chain was lengthened, indicating that the hydrophobic interaction is essential for DNA binding. It was also found that DNA binding affinity of the ligands depends on the stereochemistry of the amino group. In thermodynamic evaluation, diamino-bisTHF (3:RR8) showed a high affinity to the 12 bp duplex at a molar ratio of 1:1.     

Reconstitution of apolipoprotein A-I from human high density lipoprotein with bovine brain sphingomyelin

Reconstitution of apolipoprotein A-I was found to occur readily with bovine brain sphingomyelin (BBSM), with a maximum rate occurring at a temperature of 28 degrees C, a temperature approximating the phase transition temperature for this naturally occurring phospholipid. At BBSM:A-I weight ratios of 7.5:1 or less, a single recombinant product was observed which contained three A-I molecules per particle, which had a BBSM:A-I molar ratio of 360 to 1 and which appeared in the electron microscope as a discoidal complex with a thickness of 68 A and a diameter of 217 A. By these criteria, as well as by gel filtration, this product appears very similar to that obtained by recombination of A-I with phosphatidylcholine at elevated ratios of phospholipid/protein. No evidence was found for the existence of any BBSM:A-I complexes comparable to the smaller lecithin:A-I complex containing 200-250 mol of phospholipid and two A-I molecules per complex which has been previously reported. At BBSM:A-I ratios of 15:1 (w/w), a new type of complex was observed which was discoidal by electron microscopy but possessed a larger diameter (390 A) and higher phospholipid:protein molar ratio (535:1) than has been observed previously for recombinant complexes. The BBSM:A-I complexes were found to be significantly more resistant to denaturation by guanidine hydrochloride than the dimyristoyl phosphatidylcholine:A-I recombinant complexes. It is concluded that the mechanisms of interaction between apolipoprotein A-I and either bovine brain sphingomyelin or phosphatidylcholines are similar, but that the nature of the protein-lipid interactions with BBSM are such as to produce larger and more stable complexes than are observed with the phosphatidylcholines.     

A novel protein activity mediates DNA binding of an ATR-ATRIP complex

The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.     

Expression and purification of recombinant apolipoprotein A-I Zaragoza (L144R) and formation of reconstituted HDL particles

Apolipoprotein A-I Zaragoza (L144R) (apo A-I Z), has been associated with severe hypoalphalipoproteinemia and an enhanced effect of high density lipoprotein (HDL) reverse cholesterol transport. In order to perform further studies with this protein we have optimized an expression and purification method of recombinant wild-type apo A-I and apo A-I Z and produced mimetic HDL particles with each protein. An pET-45 expression system was used to produce N-terminal His-tagged apo A-I, wild-type or mutant, in Escherichia coli BL21 (DE3) which was subsequently purified by affinity chromatography in non-denaturing conditions. HDL particles were generated via a modified sodium cholate method. Expression and purification of both proteins was verified by SDS-PAGE, MALDI-TOF MS and immunochemical procedures. Yield was 30mg of purified protein (94% purity) per liter of culture. The reconstituted HDL particles checked via non-denaturing PAGE showed high homogeneity in their size when reconstituted both with wild-type apo A-I and apo A-I Z. An optimized system for the expression and purification of wild-type apo A-I and apo A-I Z with high yield and purity grade has been achieved, in addition to their use in reconstituted HDL particles, as a basis for further studies.     

Mechanism of loading the Escherichia coli DNA polymerase III beta sliding clamp on DNA. Bona fide primer/templates preferentially trigger the gamma complex to hydrolyze ATP and load the clamp

The Escherichia coli DNA polymerase III gamma complex clamp loader assembles the ring-shaped beta sliding clamp onto DNA. The core polymerase is tethered to the template by beta, enabling processive replication of the genome. Here we investigate the DNA substrate specificity of the clamp-loading reaction by measuring the pre-steady-state kinetics of DNA binding and ATP hydrolysis using elongation-proficient and deficient primer/template DNA. The ATP-bound clamp loader binds both elongation-proficient and deficient DNA substrates either in the presence or absence of beta. However, elongation-proficient DNA preferentially triggers gamma complex to release beta onto DNA with concomitant hydrolysis of ATP. Binding to elongation-proficient DNA converts the gamma complex from a high affinity ATP-bound state to an ADP-bound state having a 10(5)-fold lower affinity for DNA. Steady-state binding assays are misleading, suggesting that gamma complex binds much more avidly to non-extendable primer/template DNA because recycling to the high affinity binding state is rate-limiting. Pre-steady-state rotational anisotropy data reveal a dynamic association-dissociation of gamma complex with extendable primer/templates leading to the diametrically opposite conclusion. The strongly favored dynamic recognition of extendable DNA does not require the presence of beta. Thus, the gamma complex uses ATP binding and hydrolysis as a mechanism for modulating its interaction with DNA in which the ATP-bound form binds with high affinity to DNA but elongation-proficient DNA substrates preferentially trigger hydrolysis of ATP and conversion to a low affinity state.     

Complex of DNA with chromatin proteins investigated by isopycnic centrifugation in metrizamide.

Complexes of mouse main band DNA with a fraction of non-histone proteins (NHP), having a high affinity for DNA, in the absence or presence of histones have been investigated by gradient centrifugation in metrizamide. Two types of complexes were formed at an input ratio of NHP to DNA between 1 and 2.5. In metrizamide gradients a majority of DNA was found in the light complex (at the density of 1.14-1.16 g/cm3) even at the very high NHP to DNA ratio. When histones were present in the reaction mixture, most of the DNA was found in the heavy complex (1.19-1.21 g/cm3). The electrophoretic profiles of the proteins recovered from the heavy and light complexes were different; some fractions of nonhistone proteins were present only in the heavy component.     

Origin of apolipoprotein A-I polymorphism in plasma

The origin and the functional significance of apo-A-I polymorphism in man has been investigated. Together with proapo-A-I (identified as A-I1 of the polymorphic series), four other isoforms are found in human plasma, namely A-I2, A-I3, A-I4, and A-I5. A-I3 is the "mature" product of proapo-A-I conversion in plasma. In this study we provide evidence that the other, more acidic, mature apo-A-I isoproteins are derived from A-I3 by a stepwise deamidation process. This conclusion is based on the following observations. 1) Incubation of A-I3 or A-I4, either free or associated with high density lipoprotein, produces a series of more acidic isoproteins corresponding to the sequence found in plasma. The conversion process fits in well with a first order reaction, and A-I3 to A-I4 conversion occurs virtually at the same rate as A-I4 to A-I5 conversion. 2) A-I3 and A-I4 have the same NH2- and C-terminal residues. 3) Formation of apo-A-I acidic isoproteins is accompanied by liberation of ammonia. In order to investigate whether deamidation of apo-A-I results in the production of forms which have different catabolism, a series of turnover studies was carried out in normal volunteers. A-I3 and A-I4 residence times in plasma were, respectively, 3.50 +/- 0.16 and 3.00 +/- 0.10 days (mean +/- S.E.; n = 3). Degradation rate of A-I3 was 8.81 +/- 0.69 mg/kg/day and that of A-I4 was 1.66 +/- 0.15 mg/kg/day (mean +/- S.E.; n = 3). Conversion of A-I3 to A-I4 and A-I4 to A-I5 occurred at the same rate in vivo as that observed in vitro. These results are consistent with the concept that A-I3 is the precursor to the other mature apo-A-I isoforms in plasma. A-I3 is the major isoform through which apo-A-I is eliminated from plasma.