Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.     

Homologous pairing and the formation of nascent synaptic intermediates between regions of duplex DNA by RecA protein

The RecA protein from E. coli gains access to duplex DNA, by nucleation from a short single-stranded gap, to form a spiral nucleoprotein filament that is capable of interaction with homologous duplex DNA. The observations described here demonstrate that any part of the nucleoprotein filament, whether it contains single- or double-stranded DNA, is capable of pairing with homologous duplex DNA. Homologous contacts between regions of duplex DNA lead to an increase in the initial rate and final extent of joint molecule formation. The experiments indicate that pairing is facilitated by the formation of nascent synaptic intermediates between duplex DNA sequences. Using chimeric form I DNA, which is incapable of forming an inter-wound or plectonemic joint with the gapped DNA due to the presence of flanking heterologous sequences, we show that these duplex-duplex pairing reactions involve extensive underwinding of the double helix.     

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.     

Intermediates in Hin-mediated DNA inversion: a role for Fis and the recombinational enhancer in the strand exchange reaction.

The site-specific inversion reaction controlling flagellin synthesis in Salmonella involves the function of three proteins: Hin, Fis and HU. The DNA substrate must be supercoiled and contain a recombinational enhancer sequence in addition to the two recombination sites. Using mutant substrates or modified reaction conditions, large amounts of complexes can be generated which are recognized by double-stranded breaks within both recombination sites upon quenching. The cleaved molecules contain 2-bp staggered cuts within the central dinucleotide of the recombination site. Hin is covalently associated with the 5' end while the protruding 3' end contains a free hydoxyl. We demonstrate that complexes generated in the presence of an active enhancer are intermediates that have advanced past the major rate limiting step(s) of the reaction. In the absence of a functional enhancer, Hin is also able to assemble and catalyze site-specific cleavages within the two recombination sites. However, these complexes are kinetically distinct from the complexes assembled with a functional enhancer and cannot generate inversion without an active enhancer. The results suggest that strand exchange leading to inversion is mediated by double-stranded cleavage of DNA at both recombination sites followed by the rotation of strands to position the DNA into the recombinant configuration. The role of the enhancer and DNA supercoiling in these reactions is discussed.     

Characterization of the DNA binding activity of stable RecA-DNA complexes. Interaction between the two DNA binding sites within RecA helical filaments

The DNA-binding, annealing and recombinational activities of purified RecA-DNA complexes stabilized by ATP gamma S (a slowly hydrolysable analog of ATP) are described. Electrophoretic analysis, DNase protection experiments and observations by electron microscopy suggest that saturated RecA complexes formed with single- or double-stranded DNA are able to accommodate an additional single strand of DNA with a stoichiometry of about one nucleotide of added single-stranded DNA per nucleotide or base-pair, respectively, of DNA resident in the complex. This strand uptake is independent of complementarity or homology between the added and resident DNA molecules. In the complex, the incoming and resident single-stranded DNA molecules are in close proximity as the two strands can anneal in case of their complementarity. Stable RecA complexes formed with single-stranded DNA bind double-stranded DNA efficiently when the added DNA is homologous to the complexed strand and then initiate a strand exchange reaction between the partner DNA molecules. Electron microscopy of the RecA-single-stranded DNA complexes associated with homologous double-stranded DNA suggests that a portion of duplex DNA is taken into the complex and placed in register with the resident single strand. Our experiments indicate that both DNA binding sites within RecA helical filaments can be occupied by either single- or double-stranded DNA. Presumably, the same first DNA binding site is used by RecA during its polymerization on single- or double-stranded DNA and the second DNA binding site becomes available for subsequent interaction of the protein-saturated complexes with naked DNA. The way by which additional DNA is taken into RecA-DNA complexes shows co-operative character and this helps to explain how topological problems are avoided during RecA-mediated homologous recombination.     

The formation of reactive intermediate(s) of glucose 6-phosphate and lysine capable of rapidly reacting with DNA

Glucose has been shown to react nonenzymatically in vitro with DNA, to form products with spectral properties similar to those observed with the nonenzymatic glycosylation of proteins in vivo. The incubation in vitro of glucose or glucose 6-phosphate with f1 phage DNA results in a time- and concentration-dependent loss of transfection efficiency. It has also been shown that incubation in vitro of pBR322 DNA with glucose 6-phosphate prompts a loss in transformation capability as well as gross DNA alterations. In the present communication, we have investigated a model reaction of glucose 6-phosphate with the amino groups of lysine to form reactive intermediates which are capable of forming covalent adducts with DNA. The preincubation of glucose 6-phosphate and [3H]lysine leads to a time- and concentration-dependent formation of reactive intermediates. These intermediates, which accumulate with time, can subsequently react with single- or double-stranded DNA to form acid-stable complexes. Studies done with synthetic polynucleotides suggest low reactivity of the intermediate with thymidine. The formation of the reactive intermediates is saturated by the addition of excess unlabeled lysine. Once formed the intermediates are insensitive to the addition of aminoguanidine and to reduction by sodium borohydride. The chemical reactions between sugars and lysine reported here and the reactivity of that product with DNA provide a model for exploring the classes of DNA damage that may contribute to the loss of DNA function during aging.     

A method for measuring the number of single- and double-strand breaks introduced into DNA molecules by the action of deoxyribonucleases.

A method is described for measuring the average number of nuclease-induced single- and double-strand breaks per DNA molecule. The procedure involves measuring the weight-average molecular weight of DNase I-digested DNA under neutral and alkaline conditions. A statistical equation is used to calculate the number of breaks per single- or double-stranded DNA molecule from the respective weight-average molecular weights. Enzymatic incorporation of³²P into the 5′-OH ends of DNase I-induced breaks gave an independent measurement of the number of breaks per DNA molecule. Results obtained by the two different methods were in good agreement. In agreement with earlier reports we find that magnesium-activated DNase catalyzes a high frequency of single-strand breaks in DNA. The frequency of double-strand breaks is low, but significantly higher than can be explained by random accumulation of single-strand breaks. Our data suggest that the frequency of double-strand scission is affected by DNase-metal ion interactions.     

Bovine thymus poly(adenosine diphosphate ribose) polymerase. Physical properties and binding to DNA

Purified bovine thymus poly(adenosine diphosphate ribose) polymerase is a monomeric protein with a single polypeptide chain having a molecular weight of approximately 130,000, determined by sodium dodecyl sulfate-gel electrophoresis, analytical ultracentrifugation, and gel filtration. A high frictional ratio (1.81) indicated that the molecule has an elongated shape, or a high solvation, or both. The enzyme is a basic protein (pI 9.8), and amino acid analysis showed a relatively high lysine content. The enzyme activity is dependent on double-stranded DNA and is solely correlated with single- or double-stranded breaks on the DNA. Filter binding assay technique showed that the enzyme-activating efficiency of DNA correlated sufficiently with its enzyme-binding efficiency. Thus, a very high enzyme-activating efficiency of a DNA fraction (active DNA) which was separated from the crude enzyme fraction is mainly due to its high enzyme-binding efficiency. It was also shown that single-stranded DNA and heparin had a strong inhibitory effect on the binding of the enzyme to double-stranded DNA, whereas competitive inhibitors did not affect the binding, We interpret these results to indicate that the binding of the enzyme to double-stranded DNA is a prerequisite step to its catalytic activity and has a dual function: (a) to position the enzyme on specific binding sites such as single- or double-stranded breaks on the DNA, and (b) to induce an active conformation of the enzyme.     

Hexameric ring structure of the full-length archaeal MCM protein complex

In eukaryotes, a family of six homologous minichromosome maintenance (MCM) proteins has a key function in ensuring that DNA replication occurs only once before cell division. Whereas all eukaryotes have six paralogues, in some Archaea a single protein forms a homomeric assembly. The complex is likely to function as a helicase during DNA replication. We have used electron microscopy to obtain a three-dimensional reconstruction of the full-length MCM from Methanobacterium thermoautotrophicum. Six monomers are arranged around a sixfold axis, generating a ring-shaped molecule with a large central cavity and lateral holes. The channel running through the molecule can easily accommodate double-stranded DNA. The crystal structure of the amino-terminal fragment of MCM and a model for the AAA+ hexamer have been docked into the map, whereas additional electron density suggests that the carboxy-terminal domain is located at the interface between the two domains. The structure suggests that the MCM complex is likely to act in a different manner to traditional hexameric helicases and is likely to bear more similarity to the SV40 large T antigen or to double-stranded DNA translocases.     

Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion

A variety of testicular insults can induce changes in the structure of spermatozoal chromatin, resulting in spermatozoal DNA that is more susceptible to acid-induced denaturation. The degree of change in the DNA can be measured using the sperm chromatin structure assay (SCSA). The SCSA measures the relative amounts of single- and double-stranded DNA after staining with the metachromatic dye, acridine orange. Here we used a stallion model (n = 4) to study the effects of scrotal heat stress on spermatozoal DNA. This model was created by insulating stallion testes for 48 h and collecting sperm daily thereafter for 60 days. Changes in the SCSA were then correlated with protamine disulfide content and protamine types and levels. Results of the SCSA indicated that the susceptibility of spermatozoal DNA to denaturation was dependent on the spermatogenic cell stage that the ejaculated sperm was in at the time of the heat stress. Spermatozoa with altered DNA had a decrease in the extent of disulfide bonding that was associated with an increase in the susceptibility of DNA to denaturation. However, there were no detectable changes in either the protamine type or level. Thus, in this model, decreased disulfide bonding is associated with an increased susceptibility of spermatozoal DNA to denaturation in the absence of protamine changes.     

Location of the disulfide bonds in human plasma prekallikrein: the presence of four novel apple domains in the amino-terminal portion of the molecule

The location of 16 of the 18 disulfide bonds in human plasma prekallikrein was determined by amino acid sequence analysis of cystinyl peptides produced by chemical and enzymatic digestions. A unique structure, named the apple domain, was established for each of the four tandem repeats in the amino-terminal portion of the molecule. The apple domains (90 or 91 amino acids) contain 3 highly conserved disulfide bonds linking the first and sixth, second and fifth, and third and fourth half-cystine residues present in each repeat. The fourth tandem repeat contains an extra disulfide bond that forms a second small loop within the apple domain. The carboxyl-terminal portion of plasma prekallikrein containing the catalytic region of the molecule was found to have disulfide bonds located in positions similar to those of other serine proteases.     

Purification and properties of an endonuclease from nuclei of uninfected and polyoma-infected 3T3 cells.

An endonuclease activity has been purified approximately 800-fold from nuclei of 3T3 cells infected with polyoma virus. The purfied enzyme catalyzes an endonucleoytic cleavage of single- and double-stranded DNA and single-stranded RNA. Evidence that the activity towards these substrates resides in the same protein molecule is provided by the finding that they co-sediment in sucrose gradients and have identical rates of heat inactivation. Studies on the DNase activity shows that the rate of hydrolysis of single-stranded T7 DNA is 100-fold greater than that for double-stranded T7 DNA. Single-stranded DNA is extensively hydrolyzed to low molecular weight acid-insoluble products. With duplex DNA as substrate, only a limited number of single strand breaks are introduced. A limit digest with polyoma DNA (component I) as substrate results in the introduction of four breaks per strand. The phosphdiester bond interruptions can be repaired by polynucleotide ligase. Approximately 80% of the 5' termini present at the point of phosphodiester bond cleavage are purine nucleotides. Additional studies have demonstrated that a similar endonuclease is present in nuclei of uninfected cells and that this enzyme purified 400-fold has catalytic properties identical with those of the endonuclease from infected cells.     

Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro.

Replication of bacteriophage phi 29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism. In the present paper we have used an in vitro phi 29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. Two types of replicative intermediates were observed: type I (full-length double-stranded phi 29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length phi 29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a single-stranded DNA portion spanning to the other end). Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro phi 29 DNA replication system. Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner. Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded phi 29 DNA molecules were synthesized. In addition, replication of recombinant phi 29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded phi 29 DNA molecules. These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge.     

Location of the disulfide bonds in human coagulation factor XI: the presence of tandem apple domains

Factor XI is a plasma glycoprotein that participates in the blood coagulation cascade. Of the 19 disulfide bonds present in each of the subunits of the human protein, 16 were determined by amino acid sequence analysis of peptide fragments produced by chemical and enzymatic digestion. Four apple domains of 90 or 91 amino acids were identified in the tandem repeats present in the amino-terminal portion of each subunit of factor XI. The disulfide bonds in the carboxyl-terminal portion of the molecule were similar to those in the catalytic region of other serine proteases. The two identical subunits of factor XI were connected by a single disulfide bond at Cys321 linking each of the fourth apple domains while each of the Cys residues at position 11 in the first apple domains forms a disulfide bond with another Cys residue.     

Base excision repair intermediates as topoisomerase II poisons

Abasic sites are the most commonly formed DNA lesions in the cell and are produced by numerous endogenous and environmental insults. In addition, they are generated by the initial step of base excision repair (BER). When located within a topoisomerase II DNA cleavage site, "intact" abasic sites act as topoisomerase II poisons and dramatically stimulate enzyme-mediated DNA scission. However, most abasic sites in cells are not intact. They exist as processed BER intermediates that contain DNA strand breaks proximal to the damaged residue. When strand breaks are located within a topoisomerase II DNA cleavage site, they create suicide substrates that are not religated readily by the enzyme and can generate permanent double-stranded DNA breaks. Consequently, the effects of processed abasic sites on DNA cleavage by human topoisomerase IIalpha were examined. Unlike substrates with intact abasic sites, model BER intermediates containing 5'- or 3'-nicked abasic sites or deoxyribosephosphate flaps were suicide substrates. Furthermore, abasic sites flanked by 5'- or 3'-nicks were potent topoisomerase II poisons, enhancing DNA scission approximately 10-fold compared with corresponding nicked oligonucleotides that lacked abasic sites. These findings suggest that topoisomerase II is able to convert processed BER intermediates to permanent double-stranded DNA breaks.     

Fluorescent properties of DNA base analogue tC upon incorporation into DNA--negligible influence of neighbouring bases on fluorescence quantum yield

The quantum yield of the fluorescent tricyclic cytosine analogue, 1,3-diaza-2-oxophenothiazine, tC, is high and virtually unaffected by incorporation into both single- and double-stranded DNA irrespective of neighbouring bases (0.17–0.24 and 0.16–0.21, respectively) and the corresponding fluorescence decay curves are all mono-exponential, properties that are unmatched by any base analogue so far. The fluorescence lifetimes increase when going from tC free in solution (3.2 ns) to single- and double-stranded DNA (on average 5.7 and 6.3 ns, respectively). The mono-exponential decays further support previous NMR results where it was found that tC has a well-defined position and geometry within the DNA helix. Furthermore, we find that the oxidation potential of tC is 0.4 V lower than for deoxyguanosine, the natural base with the lowest oxidation potential. This suggests that tC may be of interest in charge transfer studies in DNA as an electron hole acceptor. We also present a novel synthetic route to the phosphoramidite form of tC. The results presented here together with previous work show that tC is a very good C-analogue that induces minimal perturbation to the native structure of DNA. This makes tC unique as a fluorescent base analogue and is thus highly interesting in a range of applications for studying e.g. structure, dynamics and kinetics in nucleic acid systems.     

Modeling the role of disulfide bonds in protein folding: Entropic barriers and pathways

The role of disulfide bonds in directing protein folding is studied using lattice models. We find that the stability and the specificity of the disulfide bond interactions play quite different roles in the folding process: Under some conditions, the stability decreases the overall rate of folding; the specificity, however, by yielding a simpler connectivity of intermediates, always increases the rate of folding. This conclusion is intimately related to the selection mechanism entailed by entropic driving forces, such as the loop formation probability, and entropic barriers separating the native and the many native-like metastable states. The folding time is found to be a minimum for a certain range of the effective disulfide bond interaction. Examination of a model, which allows for the formation of disulfide bonded intermediates, suggests that folding proceeds via a threestage multiple pathways kinetics. We show that there are pathways to the native state involving only native-like intermediates, as well as those that are mediated by nonnative intermediates. These findings are interpreted in terms of the appropriate energy landscape describing the barriers connecting low energy conformations. The consistency of our conclusions with several experimental studies is also discussed. © 1995 Wiley-Liss, Inc.     

Networks of DNA and RecA protein are intermediates in homologous pairing

Partial coating of single-stranded DNA by recA protein causes its aggregation, but conditions that promote complete coating inhibit independent aggregation of single strands and, instead, cause the mutually dependent conjunction of single- and double-stranded DNA in complexes that sediment at more than 10 000 S. This coaggregation is independent of homology but otherwise shares key properties of homologous pairing of single strands with duplex DNA: both processes require ATP, MgCl2, and stoichiometric amounts of recA protein; both are very sensitive to inhibition by salt and ADP. Coaggregates are closed domains that are intermediates in homologous pairing: they form faster than joint molecules, they include virtually all of the DNA in the reaction mixture, and they yield joint molecules nearly an order of magnitude faster than they exchange DNA molecules with the surrounding solution. The independent aggregation of single-stranded DNA differs in all respects except the requirement for Mg2+, and its properties correlate instead with those associated with the renaturation of complementary single strands by recA protein.     

Double-stranded DNA binding domain of poly(ADP-ribose) polymerase-1 and molecular insight into the regulation of its activity

Poly(ADP-ribose) polymerase-1 (PARP-1) modifies various proteins, including itself, with ADP-ribose polymers (automodification). Polymer synthesis is triggered by binding of its zinc finger 1 (Zn1) and 2 (Zn2) to DNA breaks and is followed by inactivation through automodification. The multiple functional domains of PARP-1 appear to regulate activation and automodification-mediated inactivation of PARP-1. However, the roles of these domains in activation-inactivation processes are not well understood. Our results suggest that Zn1, Zn2, and a domain identified in this study, the double-stranded DNA binding (DsDB) domain, are involved in DNA break-dependent activation of PARP-1. We found that binding of the DsDB domain to double-stranded DNA and DNA break recognition by Zn1 and Zn2, whose actual binding targets are likely to be single-stranded DNA, lead to the activation of PARP-1. In turn, the displacement of single- and double-stranded DNA from Zn2 and the DsDB domain caused by ADP-ribose polymer synthesis results in the dissociation of PARP-1 from DNA breaks and thus its inactivation. We also found that the WGR domain is one of the domains involved in the RNA-dependent activation of PARP-1. Furthermore, because zinc finger 3 (Zn3) has the ability to bind to single-stranded RNA, it may have an indirect role in RNA-dependent activation. PARP-1 functional domains, which are involved in oligonucleic acid binding, therefore coordinately regulate PARP-1 activity depending on the status of the neighboring oligonucleic acids. Based on these results, we proposed a model for the regulation of PARP-1 activity.     

A Mathematical Model for Basepair Opening in a DNA Double Helix

In this paper, we consider a mathematical model that draws an analogy between a DNA molecule and a mechanical system consisting of two chains of interconnected pendulums. This model is designed to explore the dynamics of the system determined by rotational movements of nucleobases around a double-stranded pentose phosphate backbone. The workability of this model is assessed with respect to various factors: inhomogeneity of the chain of nucleobases, the properties of bonds in complementary pairs, and the formation of open states. It has been shown that simplified models for averaging the characteristics of the chain of nucleobases or simplification of the type of hydrogen bond in their complementary pairs influence the type of solution significantly, impairing the validity of the results. Therefore, the approach to the solution of rotational DNA molecule dynamics developed here is more consistent with its actual biomechanics. It is shown that the emergence of open states within nucleobase pairs and restoration of the closed structure may occur in the tested mathematical model.