A kinetic proofreading mechanism for disentanglement of DNA by topoisomerases.

Cells must remove all entanglements between their replicated chromosomal DNAs to segregate them during cell division. Entanglement removal is done by ATP-driven enzymes that pass DNA strands through one another, called type II topoisomerases. In vitro, some type II topoisomerases can reduce entanglements much more than expected, given the assumption that they pass DNA segments through one another in a random way. These type II topoisomerases (of less than 10 nm in diameter) thus use ATP hydrolysis to sense and remove entanglements spread along flexible DNA strands of up to 3,000 nm long. Here we propose a mechanism for this, based on the higher rate of collisions along entangled DNA strands, relative to collision rates on disentangled DNA strands. We show theoretically that if a type II topoisomerase requires an initial 'activating' collision before a second strand-passing collision, the probability of entanglement may be reduced to experimentally observed levels. This proposed two-collision reaction is similar to 'kinetic proofreading' models of molecular recognition.     

Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells

DNA topoisomerases have been proposed to function in a variety of genetic processes in both prokaryotes and eukaryotes. Here, we have assessed the role of DNA topoisomerase II in mammalian DNA replication by determining the proximity of newly synthesized DNA to covalent enzyme-DNA complexes generated by treating cultured rat prostatic adenocarcinoma cells with teniposide. Teniposide (VM-26), an epipodophyllotoxin, is known to interact with mammalian DNA topoisomerase II so as to trap the enzyme in a covalent complex with DNA. We have found that the teniposide-induced trapping of such complexes requires MgCl2, is stimulated by ATP and is inhibited by novobiocin. The formation of covalent complexes seems to be reversible on removal of teniposide. Furthermore, analysis of the covalent complexes formed between 3H-thymidine pulse-labelled DNA and topoisomerase II following teniposide treatment reveals a direct association of the enzyme with nascent DNA fragments. Our results suggest that DNA topoisomerase II may interact with newly replicated daughter DNA molecules near DNA replication forks in mammalian cells.     

Single-molecule analysis of DNA uncoiling by a type II topoisomerase

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment. This enables them to untangle DNA and relax the interwound supercoils (plectonemes) that arise in twisted DNA. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the damping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.     

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.     

A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex

Lipases are hydrolytic enzymes which break down triacylglycerides into free fatty acids and glycerols. They have been classified as serine hydrolases owing to their inhibition by diethyl p-nitrophenyl phosphate. Lipase activity is greatly increased at the lipid-water interface, a phenomenon known as interfacial activation. X-ray analysis has revealed the atomic structures of two triacylglycerol lipases, unrelated in sequence: the human pancreatic lipase (hPL)4, and an enzyme isolated from the fungus Rhizomucor (formerly Mucor) miehei (RmL). In both enzymes the active centres contain structurally analogous Asp-His-Ser triads (characteristic of serine proteinases), which are buried completely beneath a short helical segment, or 'lid'. Here we present the crystal structure (at 3 A resolution) of a complex of R. miehei lipase with n-hexylphosphonate ethyl ester in which the enzyme's active site is exposed by the movement of the helical lid. This movement also increases the nonpolarity of the surface surrounding the catalytic site. We propose that the structure of the enzyme in this complex is equivalent to the activated state generated by the oil-water interface.     

Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA.

The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.     

核糖核苷酸还原酶研究

核糖核苷酸还原酶广泛存在于各种生物中,是生物体内唯一的催化4种核糖核苷酸还原、生成相应的脱氧核糖核苷酸的酶。该酶是DNA合成和修复的关键酶和限速酶,对细胞的增殖和分化起着调控作用。不同生物中的RR根据其结合的金属辅助因子不同而分类。虽然不同类型RR之间的氨基酸序列相似性很低,但它们有十分相似的三级结构的活性中心和相同的催化功能。RR分子中包含2个变构位点,即酶活性中心和底物特异结合位点。活性中心通过生物有机自由基的作用催化核糖核苷酸还原;底物特异结合位点通过变构作用调控4种dNTPs在细胞内的平衡。因此,该酶不仅是研究DNA合成与修复、细胞增殖与分化及癌症的治疗与抗癌药物开发的重要靶点,同时也是研究酶的结构与功能以及酶的催化机理等的重要工具。本文总结了该酶的种类与分布、结构特征、催化机理及作为抗癌药物开发靶点等方面的研究进展。     

Preferential relaxation of supercoiled DNA containing a hexadecameric recognition sequence for topoisomerase I

In prokaryotes, the degree of supercoiling of DNA can profoundly influence the use of specific promoters. In eukaryotes, a variety of indirect observations suggest that DNA topology has a similar importance in proper gene expression. Much attention has therefore been focused on the cellular proteins that control DNA supercoiling, among which are the enzymes topoisomerase I and II. A hexadecameric sequence functions as a strong attraction site for topoisomerase I. Here we report that the interaction of topoisomerase I with this sequence motif is highly specific, because a single base-pair substitution prevents strand cleavage and thereby catalytic activity at the sequence. Thus, supercoiled DNA containing the recognition sequence is relaxed preferentially by topoisomerase I compared to a control, but no difference in the relaxation rate is observed for supercoiled DNA carrying the mutated sequence. The preference for the recognition sequence seems to be an intrinsic property of all eukaryotic type I topoisomerases, suggesting that the interaction might be important in a fundamental biological process.     

Uracil-DNA glycosylase acts by substrate autocatalysis

In humans, uracil appears in DNA at the rate of several hundred bases per cell each day as a result of misincorporation of deoxyuridine (dU) or deamination of cytosine. Four enzymes that catalyse the hydrolysis of the glycosylic bond of dU in DNA to yield an apyridiminic site as the first step in base excision repair have been identified in the human genome. The most efficient and well characterized of these uracil-DNA glycosylases is UDG (also known as UNG and present in almost all known organisms), which excises U from single- or double-stranded DNA and is associated with DNA replication forks. We used a hybrid quantum-mechanical/molecular-mechanical (QM/MM) approach to determine the mechanism of catalysis by UDG. In contrast to the concerted associative mechanism proposed initially, we show here that the reaction proceeds in a stepwise dissociative manner. Cleavage of the glycosylic bond yields an intermediate comprising an oxocarbenium cation and a uracilate anion. Subsequent attack by a water molecule and transfer of a proton to D145 result in the products. Surprisingly, the primary contribution to lowering the activation energy comes from the substrate, rather than from the enzyme. This 'autocatalysis' derives from the burial and positioning of four phosphate groups that stabilize the rate-determining transition state. The importance of these phosphates explains the residual activity observed for mutants that lack key residues. A corresponding catalytic mechanism could apply to the DNA glycosylases TDG and SMUG1, which belong to the same structural superfamily as UDG.     

Intrinsic dynamics of an enzyme underlies catalysis

A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis. The concept of conformational substates has been described; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis-trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.     

内生枯草芽孢杆菌HD-1β-甘露聚糖酶基因的克隆及结构生物学分析

采用PCR技术从一株内生枯草芽孢杆菌HD-1基因组DNA中扩增出β-甘露聚糖酶基因核苷酸编码序列.序列分析表明,该基因全长1 089bp,编码362个氨基酸和一个终止密码子,N端前27个氨基酸为其信号肽.该酶氨基酸序列与相同来源的β-甘露糖苷酶同源性最高达98.34%,具有ManB保守结构域,属于糖苷水解酶家族26的一员.通过对该酶分子三维结构的预测分析,该酶的催化域形成TIM桶状结构,Cys92和Cys112形成二硫键,它们之间的部分构成该酶的氧化还原反应的活性中心,Glu100和Glu292分别为该酶的酸碱催化位点和亲核催化位点.三维结构的分析为提高酶的催化活性的和功能方面的定向改造提供了依据.     

两类脂肪酸合酶中酮酰还原酶的结构、功能和药物开发前景

自然界中脂肪酸合酶（FAS）以FASⅠ和FASⅡ两种形式存在。存在于动物中的FASⅠ是由多活性中心构成的复合酶,存在于植物和细菌中的FASⅡ则是由多个独立蛋白构成的酶体系。FASⅠ和FASⅡ中催化酮酰基团还原的酶有一定同源性,酶催化动力学和酶抑制动力学的性质相似,因分子量过大,目前尚无详细的FASⅠ空间结构信息。通过比较两种类型FAS中酮酰还原酶结构与功能的异同,进一步了解FASⅠ的结构与功能。对两种不同进化程度和存在形式的同源酶进行比较研究,将有得提示复合酶的结构与功能,并得寻找新型特异性蛋白抑制剂,为新药开发提供先导化合物,具有十分重要的理论意义和应用价值。     

Convergent evolution of similar function in two structurally divergent enzymes

An example of two related enzymes that catalyse similar reactions but possess different active sites is provided by comparing the structure of Escherichia coli thioredoxin reductase with glutathione reductase. Both are dimeric enzymes that catalyse the reduction of disulphides by pyridine nucleotides through an enzyme disulphide and a flavin. Human glutathione reductase contains four structural domains within each molecule: the flavin-adenine dinucleotide (FAD)- and nicotinamide-adenine dinucleotide phosphate (NADPH)-binding domains, the 'central' domain and the C-terminal domain that provides the dimer interface and part of the active site. Although both enzymes share the same catalytic mechanism and similar tertiary structures, their active sites do not resemble each other. We have determined the crystal structure of E. coli thioredoxin reductase at 2 A resolution, and show that thioredoxin reductase lacks the domain that provides the dimer interface in glutathione reductase, and forms a completely different dimeric structure. The catalytically active disulphides are located in different domains on opposite sides of the flavin ring system. This suggests that these enzymes diverged from an ancestral nucleotide-binding protein and acquired their disulphide reductase activities independently.     

Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G

The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.     

Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3'-->5' exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a 'right hand' shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an 'open fingers' state in which the active site samples nucleotides, and a 'closed' state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of approximately 34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.     

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).