DNA binding and nuclease protection by the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus

The DNA-binding and nuclease-protection properties of the HMf histones from the hyperthermophilic archaeon Methanothermus fervidus have been shown to be consistent with the formation of nucleosome-like structures (NLS). These proteins bind to DNA molecules as short as 20 bp and form complexes that protect DNA fragments from micrococcal nuclease (MNase) digestion that are 30 bp, ∼ 60 bp and multiples of ∼ 60 bp in length. The sequences of 49 of the ∼ 60-bp DNA fragments protected from MNase digestion by HMfA have been determined and their intrinsic curvatures calculated. A circular permutation gel mobility-shift assay was used to determine directly the curvatures for five of these sequences. HMfA bound to intrinsically curved and noncurved DNAs, but exhibited a slight preference for the model curved DNA in binding competitions with a model noncurved DNA. The results obtained are consistent with the concept that the archaeal NLS is analogous, and possibly homologous, to the central core of the eukaryal nucleosome formed by a histone (H3 + H4)2 tetramer. Received: August 11, 1996 / Accepted: November 12, 1996     

人DNA 拓扑异构酶Ⅰ在毕赤酵母中的表达及发酵条件优化

为了在体外以人DNA拓扑异构酶Ⅰ(hTopoⅠ)为靶位进行抗肿瘤化合物的快速筛选,用RT-PCR法从Hela细胞中克隆了hTopoⅠ基因ORF并在毕赤酵母中首次成功表达,表达产物可分泌到发酵上清,易于制备。蛋白酶A缺陷的重组酵母(SMD-hTopoⅠ)分泌重组酶的能力比具有蛋白酶A活性的重组酵母(X33-hTopoⅠ和KM-hTopoⅠ)更高。通过发酵条件的优化,使用BMMY(pH7．25),于20℃,每隔24h补加0．5％(V/V)的甲醇和3％(V/V)的营养液,SMD-hTopoⅠ诱导72h后可表达最高的酶活力(43000u/mL),发酵上清中hTopoⅠ可达11mg/L,约占总蛋白的10％。SDS-PAGE和Westem blot分析显示,表达的hTopoⅠ为91kD蛋白,无糖基化修饰。     

The effect of intrinsic curvature on supercoiling: Predictions of elasticity theory

Elasticity theory of naturally curved rods is employed to study the effects of intrinsic curvature on the properties of the equilibrium conformations of supercoiled DNA. The results stand in sharp contrast to those obtained when the molecule is viewed as being straight in its relaxed form. Starting from very fundamental principles of the theory, we show that the torsion of an open segment with a curved duplex axis can vary when the temperature, and along with it, the intrinsic twist is changed. Conversely, an imposed helicity, such as might be associated with binding to a histone, can change the intrinsic twist. It is also shown that another consequence of the presence of naturally curved sequences is that the twist density will, in general, vary with position along the chain in all equilibrium states. Then portions of the molecule will be more or less susceptible to interaction with other agents sensitive to such a variation. Finally, some closed equilibrium global structures uniquely associated with intrinsic curvature are discussed. © 1993 John Wiley & Sons, Inc.     

Purification and characterization of human DNA topoisomerase IIIalpha.

Human topoisomerase IIIalpha (hTopo IIIalpha), the recently identified first member of the topoisomerase IA subfamily in humans, has a central domain which is highly homologous to the yeast topoisomerase III, but an overall organization closer to that of Escherichia coli DNA topoisomerase I. In order to determine the properties of hTopo IIIalpha, compared to those of other topoisomerase IA subfamily members, we purified this enzyme to near homogeneity, together with an active site-mutant Y337F. We show that hTopo IIIalpha is able to relax negatively supercoiled DNA in a distributive manner, leading to the total disappearance of the initial substrate and the appearance of intermediate topoisomers. This DNA relaxation activity is magnesium-dependent, although a low concentration of MgCl2is sufficient to obtain efficient catalysis. 32P-transfer experiments demonstrated that hTopo IIIalpha is able to cleave a single-stranded oligonucleotide and to bind covalently to the 5'-end of the cleaved DNA. Addition of 0.5 M NaCl reverses the reaction, leading to the religation of the oligo-nucleotide. Experiments utilizing several different single-stranded oligonucleotides permitted us to map several cleavage sites and to deduce a consensus sequence for DNA cleavage (CANNN downward arrow), which is different from that for other members of the Topo IA subfamily.     

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.     

Restriction enzyme digests of rapidly renaturing fragments of vaccinia virus DNA.

Vaccinia virus DNA fragments that have been denatured by alkali and then neutralized contain a fraction that rapidly reforms duplex structures. The fraction is enriched by fractionating on hydroxyapatite columns and serves as as substrate for digestion by two restriction endonucleases isolated from Hemophilus parainfluenzae, Hpa I and HPa II. The patterns obtained by gel electrophoresis of the digested fragments show the presence of three major bands after Hpa I digestion and four major bands after Hpa II digestion. The DNA that is isolated from some of these bands quickly reforms duplex regions after alkaline denaturation. The size of the DNA segments in the major bands has been estimated to be in the range of 0.44 X 10(6) to 3.2 X 10(6) daltons. The fragments which rapidly reform duplex chains after denaturation are sensitive to single-strand-specific nucleases. These results are consistent with a model of vaccinia virus DNA which has a covalent link connecting complementary chains.     

Identification and purification of a protein that stimulates the helicase activity of the Escherichia coli Rep protein

A polypeptide (Mr = 15,000) has been purified from Escherichia coli cell extracts that significantly stimulates the duplex DNA unwinding reaction catalyzed by E. coli Rep protein. The Rep helicase unwinding reaction was stimulated by as much as 20-fold, upon addition of the stimulatory protein, using either a 71-base pair or a 343-base pair partial duplex DNA molecule as a substrate. The purified Rep helicase stimulatory protein (RHSP) had no intrinsic helicase activity or ATP hydrolysis activity and did not stimulate the single-stranded DNA-dependent ATP hydrolysis reaction catalyzed by Rep protein. It is likely that RHSP stimulates the Rep helicase unwinding reaction by stoichiometric binding to single-stranded DNA. However, a specific interaction between Rep protein and RHSP cannot be ruled out, since RHSP did not stimulate the duplex DNA unwinding reactions catalyzed by E. coli helicase I or the recently discovered 75-kDa helicase. RHSP did stimulate the duplex DNA unwinding reaction catalyzed by E. coli helicase II. The identification and subsequent purification of RHSP from cell extracts demonstrates the feasibility of using direct helicase assays to purify stimulatory proteins.     

Simian virus 40 large T-antigen G-quadruplex DNA helicase inhibition by G-quadruplex DNA-interactive agents

On the basis of growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for DNA replication, the G-quadruplex DNA unwinding ability of a prototypical replicative helicase, SV40 large T-antigen (T-ag), was investigated. Here, we demonstrate that this G-quadruplex helicase activity is robust and comparable to the duplex helicase activity of T-ag. Analysis of the SV40 genome demonstrates the presence of sequences that may form intramolecular G-quadruplexes, which are the presumed natural substrates for the G-quadruplex helicase activity of T-ag. A number of G-quadruplex-interactive agents as well as new perylene diimide (PDI) derivatives have been investigated as inhibitors of both the G-quadruplex and the duplex DNA helicase activities of T-ag. A unique subset of these G-quadruplex-interactive agents inhibits the G-quadruplex DNA unwinding activity of T-ag, relative to those reported to inhibit G-quadruplex DNA unwinding by RecQ-family helicases. We also find that certain PDIs are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic G-quadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication.     

Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor

The suicidal inactivation of Escherichia coli DNA polymerase I by epoxy-ATP has been previously reported (Abboud et al., 1978). We have examined in detail the mechanism of this inactivation utilizing a synthetic DNA template-primer of defined sequence. Epoxy-ATP inactivates the large fragment of DNA polymerase I (the Klenow fragment) in a time- and concentration-dependent manner (KI = 21 microM; kinact = 0.021 s-1). Concomitant with inactivation is the incorporation of epoxy-AMP into the primer strand. The elongated DNA duplex directly inhibits the polymerase activity of the enzyme (no time dependence) and is resistant to degradation by the 3'----5' exonuclease and pyrophosphorylase activities of the enzyme. Inactivation of the enzyme results from slow (4 X 10(-4) s-1) dissociation of the intact epoxy-terminated template-primer from the enzyme and is thus characterized as a tight-binding inhibition. Surprisingly, while the polymerase activity of the enzyme is completely suppressed by epoxy-ATP, the 3'----5' exonuclease activity remains intact. The data presented demonstrate that even though the polymerase site is occupied with duplex DNA, the enzyme can bind a second DNA duplex and carry out exonucleolytic cleavage.     

RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses I. Directing Influence of DNA in the Reaction

The template requirements and deoxyribonucleic acid (DNA) products of the DNA polymerases isolated from Rauscher leukemia and avian myeloblastosis viruses have been examined. All DNA preparations or synthetic polydeoxynucleotides which are active as primers possess a duplex structure containing single-stranded regions with a 3'-hydroxyl terminus. Native DNA and fully single-stranded DNA are inactive; moreover, their activity is not enhanced by sonic oscillation or treatment with micrococcal nuclease, Neurospora nuclease, or low levels of deoxyribonuclease I. Poor DNA templates are activated by treatment with exonuclease III, large amounts of deoxyribonuclease I, or an endonuclease isolated from Rauscher viral preparations. In reactions primed with deoxyadenylate-deoxythymidylate copolymer, the product formed is covalently attached to primer strands, indicating that no new strands are initiated. DNA polymerase products formed with exonuclease III- or deoxyribonuclase I-treated DNA are duplex structures. Short single-stranded regions are completely filled in, whereas long single-stranded regions are only partly repaired. DNA preparations containing extensive single-stranded regions are poorly utilized as templates.     

An endonuclease activity of venom phosphodiesterase specific for single-stranded and superhelical DNA.

A homogeneous preparation of venom phosphodiesterase from Crotalus adamanteus possesses an intrinsic endonuclease activity, specific for superhelical (form I) and single-stranded DNA. The phosphodiesterase degrades single-stranded T7 DNA by endonucleolytic cleavages. Duplex T7 DNA is hydrolyzed by the liberation of acid-soluble products simultaneously from the 3' and 5' termini but without demonstrable internal scissions in duplex regions. Since venom phosphodiesterase is known to hydrolyze oligonucleotides stepwise from the 3' termini, the cleavage at the 5' end of duplex T7 DNA is ascribed to an endonuclease activity. Form I PM2 DNA is nicked to yield first relaxed circles and then linear DNA which is subsequently hydrolyzed only from the chain termini. The linear duplex DNA intermediates consist of a discrete series of fragments (11 are usually resolved on agarose gels) with initial molecular weights ranging from 6.3 x 10(6) (the intact PM2 DNA size) to approximately 1 x 10(6). The cleavage of the form I molecule must, therefore, occur at a limited number of unique sites. The enzyme also cleaves nonsuperhelical, covalently closed circular PM2 DNA but at a 10(4) times slower rate. Both the endonuclease activity on form I DNA and the known exonuclease activity co-migrate on polyacrtkanude gels, are optimally active at pH 9, are stimulated by small concentrations of Mg2+, and are similarly inactivated by heat, reducing agents, and EDTA.     

Effect of LNA- and OMeN-modified oligonucleotide probes on the stability and discrimination of mismatched base pairs of duplexes

Locked nucleic acid (LNA) and 2'-O-methyl nucleotide (OMeN) are the most extensively studied nucleotide analogues. Although both LNA and OMeN are characterized by the C3'-endo sugar pucker conformation, which is dominant in A-form DNA and RNA nucleotides, they demonstrate different binding behaviours. Previous studies have focused attention on their properties of duplex stabilities, hybridization kinetics and resistance against nuclease digestion; however, their ability to discriminate mismatched hybridizations has been explored much less. In this study, LNA- and OMeN-modified oligonucleotide probes have been prepared and their effects on the DNA duplex stability have been examined: LNA modifications can enhance the duplex stability, whereas OMeN modifications reduce the duplex stability. Next, we studied how the LNA:DNA and OMeN:DNA mismatches reduced the duplex stability. Melting temperature measurement showed that different LNA:DNA or OMeN:DNA mismatches indeed influence the duplex stability differently. LNA purines can discriminate LNA:DNA mismatches more effectively than LNA pyrimidines as well as DNA nucleotides. Furthermore, we designed five LNA- and five OMeN-modified oligonucleotide probes to simulate realistic situations where target-probe duplexes contain a complementary LNA:DNA or OMeN:DNA base pairs and a DNA:DNA mismatch simultaneously. The measured collective effect showed that the duplex stability was enhanced by the complementary LNA:DNA base pair but decreased by the DNA:DNA mismatch in a position-dependent manner regardless of the chemical identity and position of the complementary LNA:DNA base pair. On the other hand, the OMeN-modified probes also showed that the duplex stability was reduced by both the OMeN modification and the OMeN:DNA mismatch in a position-dependent manner.     

Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.     

X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution.

The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.     

NF-kappa B binding mechanism: a nuclear magnetic resonance and modeling study of a GGG --> CTC mutation

We present the solution structure of the nonpalindromic 16 bp DNA 5'd(CTGCTCACTTTCCAGG)3'. 5'd(CCTGGAAAGTGAGCAG)3' containing a mutated kappaB site for which the mutation of a highly conserved GGG tract of the native kappaB HIV-1 site to CTC abolishes NF-kappaB binding. 1H and 31P NMR spectroscopies have been used together with molecular modeling to determine the fine structure of the duplex. NMR data show evidence for a BI-BII equilibrium of the CpA.TpG steps at the 3'-end of the oligomer. Models for the extreme conformations reached by the mutated duplex (denoted 16M) are proposed in agreement with the NMR data. Since the distribution of BII sites is changed in the mutated duplex compared to that of the native duplex (denoted 16N), large differences are induced in the intrinsic structural properties of both duplexes. In particular, in BII structures, 16M shows a kink located at the 3'-end of the duplex, and in contrast, 16N exhibits an intrinsic global curvature toward the major groove. Whereas 16N can reach a conformation very favorable for the interaction with NF-kappaB, 16M cannot mimic such a conformation and, moreover, its deeper and narrower major groove could hinder the DNA-protein interactions.