Circular Substrate Research Articles

Poly(ADP-ribose) Polymerase and Ku Autoantigen Form a Complex and Synergistically Bind to Matrix Attachment Sequences

Genomic sequences with a cluster of ATC sequence stretches where one strand consists exclusively of well mixed As, Ts, and Cs confer high base unpairing propensity under negative superhelical strain. Such base unpairing regions (BURs) are typically found in scaffold or matrix attachment regions (SARs/MARs) that are thought to contribute to the formation of the loop domain structure of chromatin. Several proteins, including cell type-specific proteins, have been identified that bind specifically to double-stranded BURs either in vitro or in vivo. By using BUR-affinity chromatography to isolate BUR-binding proteins from breast cancer SK-BR-3 cells, we almost exclusively obtained a complex of poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK). Both PARP and DNA-PK are activated by DNA strand breaks and are implicated in DNA repair, recombination, DNA replication, and transcription. In contrast to the previous notion that PARP and Ku autoantigen, the DNA-binding subunit of DNA-PK, mainly bind to free ends of DNA, here we show that both proteins individually bind BURs with high affinity and specificity in an end-independent manner using closed circular BUR-containing DNA substrates. We further demonstrate that PARP and Ku autoantigen form a molecular complex in vivo and in vitro in the absence of DNA, and as a functional consequence, their affinity to the BURs are synergistically enhanced. ADP-ribosylation of the nuclear extract abrogated the BUR binding activity of this complex. These results provide a mechanistic link toward understanding the functional overlap of PARP and DNA-PK and suggest a novel role for these proteins in the regulation of chromatin structure and function.

Epstein–Barr Virus Plasmid Model System for Analyzing Recombination in Human Cells

Homologous recombination stimulated by a double-strand break at a desired target site offers a method to achieve site-specific integration useful for gene therapy and other genetic engineering. To test parameters needed for this strategy, we developed an Epstein–Barr virus shuttle vector model system as a genetic tool. This extrachromosomal plasmid assay system has several advantages over a chromosomal assay. The system detects all classes of recombination events without selection and allows rapid analysis of the frequency and nature of recombination events. We found that a double-strand break at the target site stimulated a large increase in recombination frequency. The resulting recombinants included one-sided insertion events, as well as two-sided or gene conversion events. A circular donor substrate was more effective in recombination than linearized donor DNA.

Biochemical characterization of adeno-associated virus rep68 DNA helicase and ATPase activities.

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45 degreesC, and an optimal divalent-cation concentration of 5 mM MgCl2. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The Km values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 microM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3'-to-5' direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5' or 3' tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3' tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.

Methanococcus jannaschii flap endonuclease: expression, purification, and substrate requirements.

The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity. FEN retained activity after preincubation at 95 degrees C+ for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5' end adjacent to the single-strand-duplex junction. Deletion of the free 3' end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3' end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5' end failed to block cleavage, although this substrate was refractory to cleavage by the 5'-3' exonuclease activity of Taq DNA polymerase. For verification, the free 5' end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for Taq DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5' single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion phiX174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.

Diverse RNA substrates for aminoacylation: clues to origins?

The aminoacylation of RNA structures generally is considered the starting point for the emergence of the theater of proteins from the RNA world. Once an aminoacyl ester linkage is established with an RNA acceptor, peptide bond formation is thermodynamically favored. In contemporary protein biosynthesis, aminoacylation is directed to the 3′-end of the tRNA structure, which, in turn, brings the activated amino acid to the ribosomes for addition to the growing polypeptide chain. But during the last 9 years a variety of artificial RNA substrates for aminoacylation has been reported (1–8). And in this issue of the Proceedings, the list of examples is expanded in a significant way. Felden and Giege (9) describe for the first time an aminoacylation system based on a circular RNA substrate. The results obtained with this novel substrate add further support to the idea that the contemporary system of tRNA aminoacylation could have grown out of early aminoacylation systems that used diverse RNA oligonucleotide substrates. Interactions between some of these aminoacylated substrates could have led to a primitive system of peptide synthesis.

Coated with a Thick Thermal Barrier Coating Thermal Shock Testing of Burner Cans

Thick (1.8 mm) thermal barrier coatings were air-plasma-sprayed onto two different substrate geometries, including small circular substrates and burner cans. Two different top-coating spray parameters were used, where the settings of the substrate temperature and the lamella thickness were varied. A segmentation crack network was found in the top coatings sprayed using a high substrate temperature and a high lamella thickness. The density of segmentation cracks was found to be independent of substrate geometry. No segmentation cracks were found in the top-coatings when a low substrate temperature and a low lamella thickness were used. In the segmented burner can, after 1000 thermal shock cycles, the segmentation crack network was still stable and no severe cracks had formed in the top coating. In the nonsegmented burner can, cracks were formed after only 35 thermal shock cycles. Among the crack types, horizontally oriented cracks were found in the top coating close to, and sometimes reaching, the bond coating. Cracks of this type are not tolerated in thermal barrier coatings because they can cause failure of the coating. Regarding the lifetime of the segmented burner can, it is believed the failure will be dependent on other mechanisms, such as bond-coating oxidation or top-coating decomposition.

Circular substrates of the hammerhead ribozyme shift the internal equilibrium further toward cleavage.

To test whether the Y-shaped conformation of the hammerhead ribozyme is maintained throughout the catalytic pathway, the cleavage properties of circular substrates which bind the ribozyme through helices I and II were determined. Constraining the position of helices I and II in this manner did not significantly alter the rate constant for cleavage, consistent with no large rearrangement of the helices occurring during catalysis. Unexpectedly, the "internal" equilibrium between the cleavage and ligation reactions for the circular hammerheads was shifted further toward cleavage. This effect was due to the rate of ligation of the circular substrate being slower than the corresponding linear substrate. The temperature dependence of the internal equilibrium of the circular substrate revealed that although restricting the flexibility of the hammerhead reduced the favorable entropy change associated with cleavage as expected, the unfavorable enthalpy change was reduced as well, resulting in greater overall cleavage.

The post-incision steps of the DNA base excision repair pathway in Escherichia coli: studies with a closed circular DNA substrate containing a single U:G base pair.

The DNA base excision repair pathway is responsible for removal of oxidative and endogenous DNA base damage in both prokaryotes and eukaryotes. This pathway involves formation of an apurinic/apyrimidinic (AP) site in the DNA, which is further processed to restore the integrity of the DNA. In Escherichia coli it has been suggested that the major mode of repair involves replacement of a single nucleotide at the AP site, based on repair synthesis studies using oligonucleotide substrates containing a unique uracil base. The mechanism of the post-incision steps of the bacterial base excision repair pathway was examined using a DNA plasmid substrate containing a single U:G base pair. Repair synthesis carried out by repair-proficient ung, recJ and xon E.coli cell extracts was analyzed by restriction endonuclease cleavage of the DNA containing the uracil lesion. It was found that replacement of the uracil base was always accompanied by replacement of several nucleotides ( approximately 15) 3' of the uracil and this process was absolutely dependent on initial removal of the uracil base by the action of uracil-DNA glycosylase. In contrast to findings with oligonucleotide substrates, replacement of just a single nucleotide at the lesion site was not detected. These results suggest that repair patch length may be substrate dependent and a re-evaluation of the post-incision steps of base excision repair is suggested.

Periodic strips on planar and circular cylindrical substrates: Exact and asymptotic analysis

Periodic strips on planar and circular cylindrical substrates: Exact and asymptotic analysis

Impairment of Proliferating Cell Nuclear Antigen-dependent Apurinic/Apyrimidinic Site Repair on Linear DNA

Repair of apurinic/apyrimidinic (AP) sites by mammalian cell extracts was compared using circular and linear DNA substrates. Extracts prepared from DNA polymerase beta (polbeta)-proficient mouse fibroblasts repaired AP sites on both circular and linear DNA. However, extracts from the isogenic polbeta-knockout cells repaired AP sites on circular DNA but not efficiently on linear DNA. The circularity-dependent repair by the polbeta-knockout cell extract was completely inhibited by anti-proliferating cell nuclear antigen (PCNA) antibody but fully restored by addition of purified PCNA. Pretreatment of the linear DNA with AP endonuclease did not improve repair, indicating that impairment of AP site repair on linear DNA by polbeta-knockout cell extracts is not due to inefficiency of damage incision but rather to deficiency at the subsequent steps. These results indicate that AP sites can be repaired on circular DNA by the PCNA-dependent pathway in addition to the polbeta-dependent pathway and that the PCNA-dependent repair mechanism is poorly functional on linear DNA in vitro.

Nitrogen Defect Concentration in Chemical Vapor Deposited Homoepitaxial Diamond at High TemperatureS

AbstractHomoepitaxial diamond films were grown on polished single crystal {100} oriented natural type Ila diamond circular substrates using high density microwave plasma chemical vapor deposition (MPCVD). Twelve homoepitaxial diamond films were grown under a variety of substrate temperatures (1000 C-2000 C), methane concentration (1% - 3% in hydrogen gas) and processing pressure(60 - 150 torr). Electron paramagnetic resonance (EPR) studies demonstrate that nitrogen is largely incorporated as singly substitutional impurity (P1-center) and nitrogen's concentration is in the range of 10 -100 parts per million. Nitrogen's concentration also shows a systematic decrease with increasing substrate temperatures. The temperature variation of nitrogen at high temperatures is discussed within a model where single substitutional nitrogen aggregates to form nitrogen defects which are not EPR active.

The ParB protein encoded by the RP4 par region is a Ca2+-dependent nuclease linearizing circular DNA substrates

The parCBA operon, which together with the parDE operon constitutes an efficient stabilization system of the broad-host-range plasmid RP4, encodes a 20 kDa polypeptide (ParB), which exhibits sequence homology to nucleases. The ParB protein was overexpressed by means of an inducible tac-promoter system. Plate assays with herring sperm DNA as substrate provided evidence for nuclease activity. The ParB nuclease shows specificity for circular DNA substrates and linearizes them regardless of the presence in cis of parts of the RP4 partitioning region. The nuclease activity in vitro is stimulated by the presence of Ca2+ ions. EDTA (5 mM) completely inhibits nuclease activity. By restriction analysis of the ParB-linearized products, cleavage of circular DNA substrates taking place preferentially at specific sites was demonstrated. Run-off sequencing and primer extension analysis of ParB-linearized plasmid DNA revealed a specific target for ParB action adjacent to an AT-rich region containing palindromic sequence elements on a pBR322-derived plasmid.

Influence of the substrate surface on ion concentrations in a large volume microwave plasma

The influence of different substrates on the ion concentration distributions in the large volume slot antenna microwave plasma source SLAN II was examined. The plasma chamber of SLAN II is formed by a quartz tube of 66 cm in diameter and 44 cm in length. Microwave power of 4 kW at 2.45 GHz is fed through 30 resonant coupling slots from an annular cavity. Ion concentration and electron temperature distributions were measured by use of a double Langmuir probe. The pressures of both argon and oxygen were changed from 1 Pa to 5 Pa. Axial ion concentration distributions were measured with and without circular substrates of 15 cm diameter, made of different materials (aluminum, stainless steel, glass) and placed in the center of the source perpendicular to its axis. The ion concentration at a position 2 mm from from the substrate surface is reduced by a factor of 2. in comparison with the plasma without substrate. Substrates cause a slight increase of electron temperature. The influence of the substrate on the ion concentration distribution is enhanced with increasing pressure and is stronger for argon than for oxygen. Only slight differences between plasma parameter distributions for different substrate materials are observed. The influence of axially positioned substrates on radial ion concentration distributions was also examined. In this case a weaker influence of the substrate on the ion concentration distribution is observed because of the vicinity of the direct power coupling zone (quartz tube).

Characterization of a helicase, DHEL III, from Drosophila melanogaster embryonic nuclei.

Nuclear extracts prepared from Drosophila preblastoderm embryos contain a nucleoside 5'-triphosphate-dependent helicase that unwinds circular or linear partial duplex DNA substrates and moves along the DNA in 3' to 5' direction. The helicase reaction is supported with both nucleoside and deoxynucleoside 5'-triphosphates and requires divalent cations. Optimum activity in vitro is achieved with dATP or ATP and with MgCl2. In glycerol density gradients, embryonic enzyme migrates as a single peak of around 90kDa and it is the most prominent DNA-unwinding activity detectable in nuclear extracts prepared from embryos 0-2 hours after egg laying. In embryos collected 2-4 hours after egg laying this DNA unwinding activity increases and then gradually decreases in embryos collected 4-8 and 8-16 hours after egg laying.

Selection of non-specific DNA cleavage sites by the type IC restriction endonuclease EcoR124I

The Type IC restriction endonuclease EcoR124I binds specifically to its recognition sequence but subsequently translocates non-specific DNA past the complex in an ATP-dependent mechanism. The enzyme thus has the potential to cleave DNA at loci distant from the recognition site. We have scrutinised the link between translocation and cleavage on linear and circular DNA substrates. On linear DNA carrying two recognition sites, the majority of cleavages at loci distant from the recognition site occurred between the two sites, regardless of the inter-site distance or relative orientations. On circular DNA carrying one site, distant cleavages occurred throughout the DNA but an equivalent linear molecule underwent considerably fewer cleavages at distant loci. These results agree with published models for DNA tracking. However, on every molecule investigated, discrete cleavage sites were also observed within ±250 bp of the recognition sites. The localised cleavages were not confined to particular DNA sequences and were independent of DNA topology. We propose a model to account for both distant and localised cleavage events. The conformation of the DNA loop extruded during tracking may result in two DNA segments being held in proximity to the restriction moiety on the protein, one close to the EcoR124I site and another distant from the site: cleavage may occur in either segment. Alternatively, the cutting of DNA close to recognition sites may be the result of multiple nicks being generated in the expanding loop before any extensive translocation.

Capillary condensation between disks in two dimensions

Capillary condensation between two two-dimensional wetted circular substrates (disks) is studied by an effective free energy description of the wetting interface. The interfacial free-energy potential is developed on the basis of the theory for the wetting of a single disk, where interfacial capillary fluctuations play a dominant role. A simple approximative analytical expression of the interfacial free energy is developed and is validated numerically. The capillary condensation is characterized by the analysis of the coverage of the condensed phase, its stability, and asymptotic behaviors. The theory can be applied to the description of flocculations in two-dimensional systems of colloids.

Analysis of Incision Sites Produced by Human Cell Extracts and Purified Proteins during Nucleotide Excision Repair of a 1,3-Intrastrand d(GpTpG)-Cisplatin Adduct

Nucleotide excision repair by mammalian enzymes removes DNA damage as part of approximately 30-mer oligonucleotides by incising phosphodiester bonds on either side of a lesion. We analyzed this dual incision reaction at a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link in a closed circular duplex DNA substrate. Incisions were formed in the DNA with human cell extracts in which DNA repair synthesis was inhibited. The nicks were mapped by restriction fragment end labeling and primer extension analysis. Principal sites of cleavage were identified at the 9th phosphodiester bond 3' to the lesion and at the 16th phosphodiester bond 5' to the lesion. The predominant product was found to be a 26-mer platinated oligonucleotide by hybridization to a 32P-labeled complementary DNA probe. Oligonucleotides were formed at the same rate as the 3' cleavage, suggesting that both incisions are made in a near-synchronous manner. There was, however, a low frequency of 5' incisions in the absence of 3' cleavage. The dual incision reaction was reconstituted using the purified mammalian proteins XPA, RPA, XPC, TFIIH, XPG, and a fraction containing ERCC1-XPF and IF7. All of these components were required in order to observe any cleavage.

Energy Coupling in Escherichia coli DNA Gyrase: the Relationship between Nucleotide Binding, Strand Passage, and DNA Supercoiling

Binding of the nonhydrolyzable ATP analogue 5'-adenylyl-beta, gamma-imidodiphosphate (ADPNP) to Escherichia coli DNA gyrase can lead to a limited noncatalytic supercoiling of DNA. Here we examine the efficiency of coupling between ADPNP binding and the change in linking number either of positively or negatively supercoiled plasmid DNA or of small DNA circles. The coupling efficiency varies from 100% (delta Lk = -2 per gyrase tetramer, a stoichiometry of 1) with positively supercoiled substrates under certain reaction conditions to an undetectably low value with moderately negatively supercoiled substrates (sigma = -0.046) or small circular substrates. Furthermore, the rate of ADPNP binding to the gyrase-DNA complex is also dependent on the topological state of the DNA; the previously observed slow binding of ADPNP to the complex of gyrase with linear DNA is accelerated 16-fold when the substrate DNA is negatively supercoiled, suggesting a functional interaction between the nucleotide-binding and DNA-binding domains which is independent of the strand-passage process. The implications for the normal ATP-dependent supercoiling reaction of the enzyme are considered and the results discussed in terms of current mechanistic models for DNA gyrase action and the possible in vivo roles of the enzyme.

Spectroscopic Demonstration of a Linkage between the Kinetics of NTP Hydrolysis and the Conformational State of the recA-Single-stranded DNA Complex

We recently constructed a mutant recA protein in which His-163 was replaced by a tryptophan residue; the [H163W]recA protein is functionally identical to the wild-type protein, and the Trp-163 side chain serves as a reporter group for the conformational transitions of the [H163W]recA-single-stranded DNA (ssDNA) complex. We have now examined the fluorescence properties of the [H163W]recA-ssDNA complex in the presence of a series of alternate nucleoside triphosphate cofactors. Under standard conditions (pH 7.5), ATP (S0.5 = 70 microM) and purine riboside triphosphate (PTP) (S0.5 = 110 microM) effect a 44% decrease in Trp-163 fluorescence and are active as cofactors for the DNA strand exchange reaction. In contrast, ITP (S0.5 = 400 microM) elicits only a 20% decrease in Trp-163 fluorescence (a level identical to that observed with the nucleoside diphosphates ADP, PDP, and IDP) and is inactive as a strand exchange cofactor. If the S0.5 (PTP) is increased to 130 microM (by increasing the pH of the reaction solution), the PTP-mediated quenching of Trp-163 fluorescence decreases to 20%, and PTP becomes inactive as a strand exchange cofactor. These results provide direct evidence for a linkage between the S0.5 value of a nucleoside triphosphate and the conformational state of the recA-ssDNA complex, with an S0.5 of 100-120 microM or lower required for stabilization of the strand exchange-active conformation.

Characterization of the nuclease activity of Drosophila Rrp1 on phosphoglycolate- and phosphate-modified DNA 3'-termini.

Drosophila Rrp1 includes a carboxy-terminal region homologous to Escherichia coli exonuclease III which is sufficient to repair both oxidative and alkylation damage to DNA. An apurinic/apyrimidinic endonuclease activity intrinsic to Rrp1 was characterized previously. In this work, the 3'-phosphodiesterase and 3'-phosphatase activities of Rrp1 are demonstrated and characterized. Phosphoglycolate- and phosphate-modified DNA 3'-termini are formed by oxygen radical induced DNA cleavage. To demonstrate the 3'-phosphodiesterase activity of Rrp1, a 3'-phosphoglycolate-terminated oligonucleotide substrate was generated by site-specific cleavage of a unique GpC dinucleotide by iron(II) bleomycin. Removal of the terminal phosphoglycolate is detected by mobility shift on a DNA sequencing gel. Rrp1 cleaves the phosphoglycolate and releases a product with a 3'-hydroxyl terminus. Phosphoglycolate is removed more readily than the 3'-terminal dGMP residue. Rrp1 phosphodiesterase activity is not inhibited by 120 mM NaCl, while the 3'-exonuclease is reduced 25-fold. Using a 3'-phosphate-terminated oligonucleotide, the phosphatase activity of Rrp1 is at least 25-fold lower than its phosphodiesterase or apurinic endonuclease, and 56-fold lower than exonuclease III activity on the identical substrate. Rrp1 3'-phosphatase is reduced 25-fold by 80 mM NaCl. These results were confirmed using an assay that measures the ability of Rrp1 to stimulate DNA synthesis on circular DNA substrates nicked by various DNA damage treatments. In that assay, Rrp1 poorly repairs 3'-phosphate-terminated nicks introduced by micrococcal nuclease. The significance of these enzymatic properties for the biological of Rrp1 is discussed.