StpA protein from Escherichia coli condenses supercoiled DNA in preference to linear DNA and protects it from digestion by DNase I and EcoKI

The nucleoid-associated protein, StpA, of Escherichia coli binds non-specifically to double-stranded DNA (dsDNA) and apparently forms bridges between adjacent segments of the DNA. Such a coating of protein on the DNA would be expected to hinder the action of nucleases. We demonstrate that StpA binding hinders dsDNA cleavage by both the non-specific endonuclease, DNase I, and by the site-specific type I restriction endonuclease, EcoKI. It requires approximately one StpA molecule per 250–300 bp of supercoiled DNA and approximately one StpA molecule per 60–100 bp on linear DNA for strong inhibition of the nucleases. These results support the role of StpA as a nucleoid-structuring protein which binds DNA segments together. The inhibition of EcoKI, which cleaves DNA at a site remote from its initial target sequence after extensive DNA translocation driven by ATP hydrolysis, suggests that these enzymes would be unable to function on chromosomal DNA even during times of DNA damage when potentially lethal, unmodified target sites occur on the chromosome. This supports a role for nucleoid-associated proteins in restriction alleviation during times of cell stress.     

Calcium protects DNase I from proteinase K: a new method for the removal of contaminating RNase from DNase I

DNase I and proteinase K are two enzymes commonly used in the purification of highly polymerized RNA. In the presence of EDTA DNase I is rapidly inactivated by proteinase K while in 10 mm Ca²⁺ DNase is totally immune to proteinase K inactivation even at protease concentrations of up to 1 mg/ml. RNase A, a common contaminant of “RNase-free” DNase was inactivated by proteinase K in the presence or absence of Ca²⁺. Treatment of DNase I with proteinase K in the presence of Ca²⁺ selectively removed RNase A activity as judged by rRNA and poly(A⁺ RNA ribosomal RNA degradation monitored by sucrose gradient centrifugation. These results suggest that (i) DNase A and proteinase K can be used together in the presence of Ca²⁺ to obtain better digestion of nucleoprotein complexes, and (ii) proteinase K treatment of Ca²⁺ DNase can be used to selectively remove contaminating RNase.     

Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics

Purple acid phosphatases (PAPs) are a group of heterovalent binuclear metalloenzymes that catalyze the hydrolysis of phosphomonoesters at acidic to neutral pH. While the metal ions are essential for catalysis, their precise roles are not fully understood. Here, the Fe(III)Ni(II) derivative of pig PAP (uteroferrin) was generated and its properties were compared with those of the native Fe(III)Fe(II) enzyme. The k _cat of the Fe(III)Ni(II) derivative (approximately 60 s⁻¹) is approximately 20% of that of native uteroferrin, and the Ni(II) uptake is considerably faster than the reconstitution of full enzymatic activity, suggesting a slow conformational change is required to attain optimal reactivity. An analysis of the pH dependence of the catalytic properties of Fe(III)Ni(II) uteroferrin indicates that the μ-hydroxide is the likely nucleophile. Thus, the Ni(II) derivative employs a mechanism similar to that proposed for the Ga(III)Zn(II) derivative of uteroferrin, but different from that of the native enzyme, which uses a terminal Fe(III)-bound nucleophile to initiate catalysis. Binuclear Fe(III)Ni(II) biomimetics with coordination environments similar to the coordination environment of uteroferrin were generated to provide both experimental benchmarks (structural and spectroscopic) and further insight into the catalytic mechanism of hydrolysis. The data are consistent with a reaction mechanism employing an Fe(III)-bound terminal hydroxide as a nucleophile, similar to that proposed for native uteroferrin and various related isostructural biomimetics. Thus, only in the uteroferrin-catalyzed reaction are the precise details of the catalytic mechanism sensitive to the metal ion composition, illustrating the significance of the dynamic ligand environment in the protein active site for the optimization of the catalytic efficiency. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Variants of a cloned synthetic lactose operator: I. A palindromic dimer lactose operator derived from one strand of the cloned 40-base pair operator

Starting with one strand of the 40-bp synthetic operator (Sadler et al., 1978), we have constructed and cloned a 66-bp, palindromic DNA segment with the following sequence

where the horizontal arrows indicate the locations of the two 21-bp “core? operator sequences in this segment and the vertical arrow designates the dyad axis of symmetry. Upon denaturation and rapid renaturation, each strand of this fragment forms a hairpin molecule still retaining an EcoRI cohesive end. Two hairpin molecules can be joined with T4 DNA ligase to form a duplex DNA molecule having no ends (dumbbell form A). Denaturation and rapid renaturation of dumbbell A yields a mixture of two dumbbell forms: dumbbell A which is a substrate for EcoKL, and a new form, dumbbell B, which is not a substrate. Each of the conformations of this DNA fragment have been purified and all are active in binding lactose repressor in vitro.     

DNase I digestion as a tool for the quantitative evaluation of C-heterochromatic-DNA in situ.

The amount of DNA resisting the C-banding pre-treatments (C-heterochromatic-DNA) was found to account for the interspecific differences of genome size in different Primate groups. The evaluation of this parameter is therefore of great interest in cytotaxonomy. In this work, DNase I digestion was used instead of the pre-treatments C-banding, in an attempt to set up a suitable method for the quantitative evaluation of C-heterochromatic-DNA in both metaphase chromosomes and interphase chromatin. In fact DNase I is known to preferentially digest "active or potentially active" chromatin, and the highly repetitive and inactive DNA in C-heterochromatin should characteristically resist DNase I cleavage. As a model system, differently fixed mouse splenocytes were treated with DNase I for various times, and the digestion was monitored by flow cytometry after propidium iodide staining. In addition, mouse metaphase preparations from lymphocyte cultures were also digested with DNase I, and the amount of residual DNA was evaluated by static microfluorometry. Under controlled conditions of fixation, enzyme concentration, time and temperature, the same limit-digest can be obtained in both interphase nuclei and metaphases, which corresponds to the amount of residual DNA after C-banding and has a C-banding-like pattern in chromosomes.     

Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176 attenuated diphtheria toxin A chain.

DNA including the coding sequence for the A chain of the mutant diphtheria toxin tox 176 was cloned. The cloned mature A-chain coding sequence showed a G-to-A transition at nucleotide 383 as the only difference from the wild-type sequence. This resulted in replacement of the glycine at position 128 by aspartic acid in the predicted amino acid sequence. A eucaryotic cell expression plasmid, pTH1-176, was constructed in which the tox 176 A-chain coding sequence was attached to a truncated metallothionein promoter. The toxicity of this construct, compared with that of the corresponding wild-type diphtheria toxin A-chain plasmid, pTH1, was assessed after transfection into the human 293 cell line by an indirect transient expression assay (I. H. Maxwell, F. Maxwell, and L. M. Glode, Cancer Res. 46:4660-4664, 1986). For the same effect, 15- to 30-fold more pTH1-176 than pTH1 was required, a result consistent with previous in vitro estimates of the diminished activity of the tox 176 A chain. Controlled expression of the cloned tox 176 A-chain coding sequence may provide a means of eliminating specific cell populations in an organism, for which purpose the wild-type diphtheria toxin A chain might prove too toxic.     

Evidence for the direct interaction between tightly bound divalent metal ion and ATP on actin. Binding of the lambda isomers of beta gamma-bidentate CrATP to actin

Competition between Ca2+ and Mg2+ for binding to a single high affinity site on actin has been confirmed. Occupancy of this site only by either Ca2+ or Mg2+ affects the conformation of actin and its ability to form nuclei and hydrolyze ATP. G-actin binds the beta gamma-bidentate CrATP, a substitution inert analog of metal-ATP complexes, and shows a high specificity for the lambda isomers. Binding of CrATP to ADP-actin is accompanied by the dissociation of tightly bound ADP and Ca2+. CrATP-actin shows a high tendency to form nuclei, like MgATP-actin. Polymerization of CrATP-actin is accompanied by cleavage of the gamma-phosphate, but subsequent Pi release cannot occur because the product of the reaction is the stable CrADP-Pi complex. All these results support the view that the divalent metal ion tightly bound to actin interacts with the beta- and gamma-phosphates of ATP in the nucleotide site.     

Solution structure and thermodynamics of a divalent metal ion binding site in an RNA pseudoknot.

Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies. The pseudoknot causes efficient -1 ribosomal frameshifting in mouse mammary tumor virus. Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+preferentially stabilizes the pseudoknot relative to its constituent hairpins. The use of Co(NH3)63+as a substitute for Mg2+was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+or Co (NH3)63+. The structure of the pseudoknot-Co(NH3)63+complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem. Co(NH3)63+stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands. Hydrogen bonds are identified between the Co(NH3)63+protons and non-bridging phosphate oxygen atoms, 2' hydroxyl groups, and nitrogen and oxygen acceptors on the bases. The binding site is significantly different from that previously characterized in the major groove surface of tandem G.U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron. Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+as on addition of Co(NH3)63+, indicating that both ions bind at the same site. Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+and a temperature of 15 degrees C) were obtained for Co(NH3)63+and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration. An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot. These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots.     

Domain organization and movements in heavy metal ion pumps: papain digestion of CopA, a Cu+-transporting ATPase

To study domain organization and movements in the reaction cycle of heavy metal ion pumps, CopA, a bacterial Cu+-ATPase from Thermotoga maritima was cloned, overexpressed, and purified, and then subjected to limited proteolysis using papain. Stable analogs of intermediate states were generated using AMPPCP as a nonhydrolyzable ATP analog and AlFx as a phosphate analog, following conditions established for Ca2+-ATPase (SERCA1). Characteristic digestion patterns obtained for different analog intermediates show that CopA undergoes domain rearrangements very similar to those of SERCA1. Digestion sites were identified on the loops connecting the A-domain and the transmembrane helices M2 and M3 as well as on that connecting the N-terminal metal binding domain (NMBD) and the first transmembrane helix, Ma. These digestion sites were protected in the E1P.ADP and E2P analogs, whereas the M2-A-domain loop was cleaved specifically in the absence of ions to be transported, just as in SERCA1. ATPase activity was lost when the link between the NMBD and the transmembrane domain was cleaved, indicating that the NMBD plays a critical role in ATP hydrolysis in T. maritima CopA. The change in susceptibility of the loop between the NMBD and Ma helix provides evidence that the NMBD is associated to the A-domain and recruited into domain rearrangements and that the Ma helix is the counterpart of the M1 helix in SERCA1 and Mb and Mc are uniquely inserted before M2.     

Interaction of a protein with a palindromic sequence from murine rDNA increases the occurrence of amplification-dependent transformation in mouse cells

muNTS1, an element isolated from the nontranscribed spacer of murine rDNA, increases the occurrence of amplification-dependent transformation in mouse cells when integrated into plasmid DNA containing a selectable marker (Wegner, M., Zastrow, G., Klavinius, A., Schwender, S., Müller, F., Luksza, H., Hoppe, J., Wienberg, J., and Grummt, F. (1989) Nucleic Acids Res. 17, 9909-9932). In an initial attempt to dissect muNTS1 into its structural components we localized part of the transformation increasing activity to a long AT-rich stretch from the 5' region which interacts with HMG-I. Here we identify a second element on muNTS1 which also stimulates the rate of amplification-coupled transformation in cis. It is found in the 3' region of muNTS1 and contains the 11-base pair palindrome ATGGCTGCCAT. It is conserved in the otherwise strongly divergent ribosomal NTS regions from mouse, rat, and man and is also found in the origin/enhancer region of human papovavirus JC. The palindromic sequence interacts specifically with proteins from mouse cell extracts. Protein-DNA interaction was dependent on the presence of zinc ions in the extract. Point-specific mutations within the palindrome reduced protein-DNA complex formation substantially and concomitantly abolished the ability to stimulate the frequency of transformation. The binding activity was purified and shown to consist of two polypeptides with molecular masses of 70 and 73 kDa.     

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. I. The tryptic peptides from the maleylated protein.

Six tryptic peptides ranging in size from 3 to 126 residues were isolated from maleylated Fragment A of diphtheria toxin after tryptic hydrolysis. These peptides accounted for all 193 residues found by amino acid analysis. After demaleylation, the six peptides were purified by chromatography on Sephadex G-50, coupled with paper chromatography and electrophoresis, and were analyzed by various methods. The compositions and properties of the peptides are reported. Almost 70% of the residues were positioned within these peptides.     

Purified NPC1 protein. I. Binding of cholesterol and oxysterols to a 1278-amino acid membrane protein

The Niemann-Pick, Type C1 protein (NPC1) is required for the transport of lipoprotein-derived cholesterol from lysosomes to endoplasmic reticulum. The 1278-amino acid, polytopic membrane protein has not been purified, and its mechanism of action is unknown. Unexpectedly, we encountered NPC1 in a search for a membrane protein that binds 25-hydroxycholesterol (25-HC) and other oxysterols. A 25-HC-binding protein was purified more than 14,000-fold from rabbit liver membranes and identified as NPC1 by mass spectroscopy. We prepared recombinant human NPC1 and confirmed its ability to bind oxysterols, including those with a hydroxyl group on the 24, 25, or 27 positions. Hydroxyl groups on the 7, 19, or 20 positions failed to confer binding. Recombinant human NPC1 also bound [(3)H]cholesterol in a reaction inhibited by Nonidet P-40 above its critical micellar concentration. Low concentrations of unlabeled 25-HC abolished binding of [(3)H]cholesterol, but the converse was not true, i.e. unlabeled cholesterol, even at high concentrations, did not abolish binding of [(3)H]25-HC. NPC1 is not required for the known regulatory actions of oxysterols. Thus, in NPC1-deficient fibroblasts 25-HC blocked the processing of sterol regulatory element-binding proteins and activated acyl-CoA:cholesterol acyltransferase in a normal fashion. The availability of assays to measure NPC1 binding in vitro may further the understanding of ways in which oxysterols regulate intracellular lipid transport.     

Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition,catalysis, and metal ion dependence.

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.     

Visualising the kinetics of dissociation of actinomycin from individual sites in mixed sequence DNA by DNase I footprinting.

We have investigated the kinetics of dissociation of actinomycin D from DNA by a variation of the footprinting technique. Complexes of actinomycin with a radiolabelled DNA fragment (tyrT) were dissociated by addition of a large excess of unlabelled calf thymus DNA and the mixture subjected to DNase I footprinting at subsequent intervals. The rates at which the footprints disappeared varied between the different binding sites. The dissociation was temperature dependent with average time constants of 30 s, 10 mins and 2 hours at temperatures of 37 degrees C, 20 degrees C and 4 degrees C respectively. The dissociation from a DNA fragment containing the synthetic insert T9GCA9 was significantly faster, with a half-life of about 1 min at 20 degrees C. In contrast, the dissociation of distamycin was too fast to measure (< 5 s) even at 4 degrees C.     

Two enhancer elements for DNA replication of pSC101, par and a palindromic binding sequence of the Rep protein.

The minimal replication origin (ori) of the plasmid pSC101 has been previously defined as an approximately 220-bp region by using plasmids defective in the par region, which is a cis-acting determinant of plasmid stability. This ori region contains the DnaA binding sequence, three repeated sequences (iterons), and an inverted repeat (IR) element (IR-1), one of the binding sites of an initiator protein, Rep (or RepA). In the present study, we show that plasmids containing par can replicate at a nearly normal copy number in the absence of IR-1 but still require a region (the downstream region) between the third iteron and IR-1. Because par is dispensable in plasmids retaining IR-1, par and IR-1 can compensate each other for efficient replication. The region from the DnaA box to the downstream region can support DNA replication at a reduced frequency, and it is designated "core-ori." Addition of either IR-1 or par to core-ori increases the copy number of the plasmid up to a nearly normal level. However, the IR-1 element must be located downstream of the third iteron (or upstream of the rep gene) to enhance replication of the plasmid, while the par region, to which DNA gyrase can bind, functions optimally regardless of its location. Furthermore, the enhancer activity of IR-1 is dependent on the helical phase of the DNA double helix, suggesting that the Rep protein bound to IR-1 stimulates the activation of ori via its interaction with another factor or factors capable of binding to individual loci within ori.