DNA-protein cooperative binding through variable-range elastic coupling

Cooperativity plays an important role in the action of proteins bound to DNA. A simple mechanism for cooperativity, in the form of a tension-mediated interaction between proteins bound to DNA at two different locations, is proposed. These proteins are not in direct physical contact. DNA segments intercalating bound proteins are modeled as a worm-like chain, which is free to deform in two dimensions. The tension-controlled protein-protein interaction is the consequence of two effects produced by the protein binding. The first is the introduction of a bend in the host DNA and the second is the modification of the bending modulus of the DNA in the immediate vicinity of the bound protein. The interaction between two bound proteins may be either attractive or repulsive, depending on their relative orientation on the DNA. Applied tension controls both the strength and the range of protein-protein interactions in this model. Properties of the cooperative interaction are discussed, along with experimental implications.     

The structure of the complexes of DNA with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions. I. Circular dicroism spectroscopy

The mechanisms of interaction of the non-histone chromosomal protein HMGB1 and linker histone H1 with DNA have been studied using circular dichroism and absorption spectroscopy. Both of the proteins are located in the inter-nucleosomal regions of chromatin. It was demonstrated that properties of the DNA-protein complexes depend on the protein content and can not be considered as a simple summing up of the effects of individual protein components. Interaction of HMGB1 and H1 proteins is shown to be co-operative rather than competitive. Lysine-rich histone H1 facilitates the binding of the HMGB1 with DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino-acid residues in the C-terminal domain of the HMGB1 protein. The observed joint action of the and H1 proteins stimulates DNA condensation with formation of the anisotropic DNA-protein complexes with typical psi-type CD spectra. Structural organization of the complexes depends not only on the DNA-protein interactions, but also on the interaction between HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the character of interactions between the components in the triple DNA-HMGB1-H1 complex. Binding of Mn2+ ions causes the weakening of the DNA-protein interactions and strengthening the protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.     

Signaling by estrogens

By regulating activities and expression levels of key signaling molecules, estrogens control mechanisms that are responsible for crucial cellular functions. Ligand binding to estrogen receptor (ER) leads to conformational changes that regulate the receptor activity, its interaction with other proteins and DNA. In the cytoplasm, receptor interactions with kinases and scaffolding molecules regulate cell signaling cascades (extranuclear/nongenomic action). In the nucleus, estrogens control a repertoire of coregulators and other auxiliary proteins that are associated with ER, which in turn determines the nature of regulated genes and level of their expression (genomic action). The combination of genomic and nongenomic actions of estrogens ultimately confers the cell-type and tissue-type selectivity. Recent studies have revealed some important new insights into the molecular mechanisms underlying ER action, which may help to explain the functional basis of existing selective ER modulators (SERMs) and provide evidence into how ER might be selectively targeted to achieve specific therapeutic goals. In this review, we will summarize some new molecular details that relate to estrogen signaling. We will also discuss some new strategies that may potentially lead to the development of functionally selective ER modulators that can separate between the beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS as well as the "detrimental," proliferative effects in reproductive tissues and organs.     

Structure-based prediction of DNA target sites by regulatory proteins

Regulatory proteins play a critical role in controlling complex spatial and temporal patterns of gene expression in higher organism, by recognizing multiple DNA sequences and regulating multiple target genes. Increasing amounts of structural data on the protein-DNA complex provides clues for the mechanism of target recognition by regulatory proteins. The analyses of the propensities of base-amino acid interactions observed in those structural data show that there is no one-to-one correspondence in the interaction, but clear preferences exist. On the other hand, the analysis of spatial distribution of amino acids around bases shows that even those amino acids with strong base preference such as Arg with G are distributed in a wide space around bases. Thus, amino acids with many different geometries can form a similar type of interaction with bases. The redundancy and structural flexibility in the interaction suggest that there are no simple rules in the sequence recognition, and its prediction is not straightforward. However, the spatial distributions of amino acids around bases indicate a possibility that the structural data can be used to derive empirical interaction potentials between amino acids and bases. Such information extracted from structural databases has been successfully used to predict amino acid sequences that fold into particular protein structures. We surmised that the structures of protein-DNA complexes could be used to predict DNA target sites for regulatory proteins, because determining DNA sequences that bind to a particular protein structure should be similar to finding amino acid sequences that fold into a particular structure. Here we demonstrate that the structural data can be used to predict DNA target sequences for regulatory proteins. Pairwise potentials that determine the interaction between bases and amino acids were empirically derived from the structural data. These potentials were then used to examine the compatibility between DNA sequences and the protein-DNA complex structure in a combinatorial "threading" procedure. We applied this strategy to the structures of protein-DNA complexes to predict DNA binding sites recognized by regulatory proteins. To test the applicability of this method in target-site prediction, we examined the effects of cognate and noncognate binding, cooperative binding, and DNA deformation on the binding specificity, and predicted binding sites in real promoters and compared with experimental data. These results show that target binding sites for several regulatory proteins are successfully predicted, and our data suggest that this method can serve as a powerful tool for predicting multiple target sites and target genes for regulatory proteins.     

Basis for the historical stages for studying the biochemistry of vitamins. Noncoenzymatic mechanisms of vitamin B1

Biochemistry of vitamins is one of the leading trends in the fundamental researches of A. V. Palladin Institute of Biochemistry from the moment of its foundation in 1925. The Laboratory of Vitamins Biochemistry was organised in 1994, it was reorganized into the Department of Vitamins Biochemistry in 1966, and later it was renamed as the Department of Coenzymes Biochemistry. Now the investigations at the Coenzymes Biochemistry Department headed (from 1986) by G. V. Donchenko, Corr.-Member of the National Academy of Sciences of Ukraine, are directed to estimation of vitamins A, E, B1 and PP action molecular mechanisms. Investigation of specific protein-acceptors of vitamins and their biologically active derivatives is a contemporary and effective methodological approach to the estimation of some molecular mechanisms of vitamins action on cellular metabolism. Considering the challenging theoretical and practical aspects of the further fundamental investigation development in the molecular vitaminology the following items are currently being worked in the Department last time: 1. Study of some molecular mechanisms of thiamine and vitamin PP neurotropic action. These investigations are oriented to clearing some new aspects of noncoenzymic mechanism of its influence on the nervous cell functioning both in the norm and at some nervous diseases. 2. Study of some molecular mechanisms of regulation by means of fat-soluble vitamins A, E and their specific proteins-acceptors of DNA, RNA and protein biosynthesis in the nuclei and mitochondria of actively proliferous cells. These investigations are aimed to the estimation of molecular mechanisms of fat-soluble vitamins participation in the regulation of DNA-dependent synthesis of RNA, RNA-polymerase activity, mechanism of their anticancerogenous effect, vitamin E participation in the realisation of nuclear genetic information. 3. Study of intracellular protein-receptors, which take part in realisation of vitamins and their biologically active derivatives functions in the human and animals' organism. The investigations, directed to study of a role of retinol-binding proteins in exchange of the vitamin A and in biosynthesis of DNA, RNA and proteins, the role of tocopherol-binding proteins in realisation of biological action of vitamin E in cells and thiamine-binding proteins in realisation of neurotropic action of vitamin B1 are actively developed. 4. Investigation of mechanisms of antioxidizing and antiradical biological action of vitamin D3, ecdisterone and related biologically active compounds. Basing on the fundamental researches some vitamins preparations have been created, such as "Carotin-M", "Cardiovit", "Evit-1", "Soevit", "Metovit", "Caratel'ka" and others. The results of fundamental investigation of noncoenzymic thiamine function led us to elaboration of a new hypothesis about molecular mechanism of vitamin B1 neurotropic action. According to the hypothesis the thiamine high neuroactivity is a result of existence in the nervous ending a specific mobile thiamine pool and connection thiamine metabolism with nervous cell membrane potential and acetylcholine metabolism.     

Homologous recombination-mediated double-strand break repair

Exchange of DNA strands between homologous DNA molecules via recombination ensures accurate genome duplication and preservation of genome integrity. Biochemical studies have provided insights into the molecular mechanisms by which homologous recombination proteins perform these essential tasks. More recent cell biological experiments are addressing the behavior of homologous recombination proteins in cells. The challenge ahead is to uncover the relationship between the individual biochemical activities of homologous recombination proteins and their coordinated action in the context of the living cell.     

Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5"-flap strands of the branched DNA structure and possesses 5"-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides. Oligonucleotides bearing a photoreactive arylazido group at the 3"-end of the primer were synthesized in situ by the action of DNA polymerase using base-substituted photoreactive dUTP analogs as the substrates. The photoreactive group was also bound to the 5"-end phosphate group of the oligonucleotide by chemical synthesis. Interaction of FEN-1 with both 5"- and 3"-ends of the nick or with primer–template systems containing 5"- or 3"-protruding DNA strands was shown. Formation of a structure with the 5"-flap containing the photoreactive group results in decrease of the level of protein labeling caused by cleavage of the photoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeling of proteins of mouse fibroblast cell extract was performed using the radioactively labeled DNA duplex with the photoreactive group at the 3"-end and the apurine/apyrimidine site at the 5"-end of the nick. This structure is a photoreactive analog of an intermediate formed during DNA repair and was generated by the action of cell enzymes from the initial DNA duplex containing the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is shown to be one of the photolabeled proteins; this indicates possible participation of this enzyme in base excision repair.     

Condensin architecture and interaction with DNA: regulatory non-SMC subunits bind to the head of SMC heterodimer

Condensin and cohesin are two protein complexes that act as the central mediators of chromosome condensation and sister chromatid cohesion, respectively. The basic underlying mechanism of action of these complexes remained enigmatic. Direct visualization of condensin and cohesin was expected to provide hints to their mechanisms. They are composed of heterodimers of distinct structural maintenance of chromosome (SMC) proteins and other non-SMC subunits. Here, we report the first observation of the architecture of condensin and its interaction with DNA by atomic force microscopy (AFM). The purified condensin SMC heterodimer shows a head-tail structure with a single head composed of globular domains and a tail with the coiled-coil region. Unexpectedly, the condensin non-SMC trimers associate with the head of SMC heterodimers, producing a larger head with the tail. The heteropentamer is bound to DNA in a distributive fashion, whereas condensin SMC heterodimers interact with DNA as aggregates within a large DNA-protein assembly. Thus, non-SMC trimers may regulate the ATPase activity of condensin by directly interacting with the globular domains of SMC heterodimer and alter the mode of DNA interaction. A model for the action of heteropentamer is presented.     

Loading mechanisms of ring helicases at replication origins

Threading of DNA through the central channel of a replicative ring helicase is known as helicase loading, and is a pivotal event during replication initiation at replication origins. Once loaded, the helicase recruits the primase through a direct protein-protein interaction to complete the initial 'priming step' of DNA replication. Subsequent assembly of the polymerases and processivity factors completes the structure of the replisome. Two replisomes are assembled, one on each strand, and move in opposite directions to replicate the parental DNA during the 'elongation step' of DNA replication. Replicative helicases are the motor engines of replisomes powered by the conversion of chemical energy to mechanical energy through ATP binding and hydrolysis. Bidirectional loading of two ring helicases at a replication origin is achieved by strictly regulated and intricately choreographed mechanisms, often through the action of replication initiation and helicase-loader proteins. Current structural and biochemical data reveal a wide range of different helicase-loading mechanisms. Here we review advances in this area and discuss their implications.     

Biochemical analysis of the damage recognition process in nucleotide excision repair

XPA, XPC-hHR23B, RPA, and TFIIH all are the damage recognition proteins essential for the early stage of nucleotide excision repair. Nonetheless, it is not clear how these proteins work together at the damaged DNA site. To get insight into the molecular mechanism of damage recognition, we carried out a comprehensive analysis on the interaction between damage recognition proteins and their assembly on damaged DNA. XPC physically interacted with XPA, but failed to stabilize the XPA-damaged DNA complex. Instead, XPC-hHR23B was effectively displaced from the damaged DNA by the combined action of RPA and XPA. A mutant RPA lacking the XPA interaction domain failed to displace XPC-hHR23B from damaged DNA, suggesting that XPA and RPA cooperate with each other to destabilize the XPC-hHR23B-damaged DNA complex. Interestingly, the presence of hHR23B significantly increased RPA/XPA-mediated displacement of XPC from damaged DNA, suggesting that hHR23B may modulate the binding of XPC to damaged DNA. Together, our results suggest that damage recognition occurs in a multistep process such that XPC-hHR23B initiates damage recognition, which was replaced by combined action of XPA and RPA. XPA and RPA, once forming a complex at the damage site, would likely work with TFIIH, XPG, and ERCC1-XPF for dual incision.     

A study on the bacterial photo-toxicity of phenothiazinium based photosensitisers

"Comet assay" showed light activated (3.15 Jcm-2 over 30 min) phenothiazinium based photosensitisers (PhBPs) to induce photo-damage of Staphylococcus aureus DNA, as indicated by DNA "tails" between 80 and 120 microm. In general, PhBPs exhibited significant singlet oxygen yields (Phi(DeltaPhBP)>0.7), suggesting the use of type II mechanisms of photo-oxidation. However, the photodynamic action of PhBPs on DNA showed generally insignificant production of 7,8-dihydro-8-oxo-2'-deoxyguanosine, normally a major product of type II DNA photo-oxidation. These combined results show DNA to be a major site of action of PhBPs and suggest that this action may involve type II attack on a nucleoside(s) other than guanosine.     

The meaning of natural selection revisited at the molecular level

Various molecular interaction mechanisms cause biased transmission of genes. Meiotic drive is an example of strong bias, and compartmentalization of mammalian chromosomes reflects weak bias. Such biases are the results of interaction between DNA and proteins, and should be distinguished from natural selection. Separating the effects of molecular interaction from those of natural selection, however, is often very difficult. Natural selection and molecular mechanisms interact, and our understanding of how selection works requires revision.     

血管生成素相互作用蛋白质的筛选与鉴定

血管生成素（angiogenin, ANG）在肿瘤、神经退行性疾病和先天免疫过程中均发挥作用,但对其具体生理病理功能和作用机制的了解并不深入全面.蛋白质 蛋白质相互作用调控着细胞内的各个生物学过程,可以为目标蛋白质功能和机制的探索提供信息.本文利用酵母双杂交技术,分别从人心肌和肝cDNA文库中筛选了ANG的可能相互作用蛋白质.对筛选获得的20个候选蛋白质进行生物信息学分析,显示10个蛋白质含有EGF结构域;有5个蛋白质在KEGG 数据库已有记录,主要参与细胞黏附、通讯和迁移等过程.在以往的研究中,我们已经验证α 辅肌动蛋白2（α-actinin 2）、卵泡抑素（follistatin）、磷脂混杂酶1（phospholipid scramblase 1）和腓骨蛋白1（fibulin1）与ANG作用的真实性.本文的蛋白质沉降实验显示,ANG与腓骨蛋白2、3、4之间也存在相互作用.     

高迁移率族蛋白与真核基因表达调控

高迁移率族蛋白 (high mobility group protein , HMG) 是一系列的染色质相关蛋白,广泛存在于真核生物细胞中,含量丰富,因其在聚丙烯酰胺凝胶电泳中的高迁移率而得名 . HMG 蛋白家族可分为 HMGB 、 HMGA 和 HMGN 三类亚家族,各亚家族有其特征的结构域,这些结构域介导了 HMG 和 DNA 或染色质相关区域的相互作用 . 现已发现这些蛋白质具有多种重要生物学功能,其中几乎所有 HMG 都可以通过修饰、弯曲或改变染色质 /DNA 的结构,促进各种蛋白质因子形成大分子复合物来调节基因转录 .     

Structure-function relationship and regulation of two Bacillus subtilis DNA-binding proteins, HBsu and AbrB

Microorganisms use a number of small basic proteins for organization and compaction of their DNA. By their interaction with the genome, these proteins do have a profound effect on gene expression, growth behavior, and viability. It has to be distinguished between indirect effects as a consequence of the state of chromosome condensation and relaxation that influence the rate of RNA polymerase action as represented by the histone-like proteins, and direct effects by specific binding of proteins to defined DNA segments predominantly located around promoter sequences. This latter class is represented by the transition-state regulators that are involved in integrating various global stimuli and orchestrating expression of the genes under their regulation for a better adaptation to changes in growth rate. In this article we will focus on two different but abundant DNA binding proteins of the gram-positive model organism Bacillus subtilis, the histone-like HBsu as a member of the unspecific and the transition state regulator AbrB as a member of specific classes of DNA binding proteins.     

The MukB-ParC Interaction Affects the Intramolecular,Not Intermolecular,Activities of Topoisomerase IV

Proper chromosome organization is accomplished through binding of proteins such as condensins that shape the DNA and by modulation of chromosome topology by the action of topoisomerases. We found that the interaction between MukB, the bacterial condensin, and ParC, a subunit of topoisomerase IV, enhanced relaxation of negatively supercoiled DNA and knotting by topoisomerase IV, which are intramolecular DNA rearrangements but not decatenation of multiply linked DNA dimers, which is an intermolecular DNA rearrangement required for proper segregation of daughter chromosomes. MukB DNA binding and a specific chiral arrangement of the DNA was required for topoisomerase IV stimulation because relaxation of positively supercoiled DNA was unaffected. This effect could be attributed to a more effective topological reconfiguration of the negatively supercoiled compared with positively supercoiled DNA by MukB. These data suggest that the MukB-ParC interaction may play a role in chromosome organization rather than in separation of daughter chromosomes.