A comparison of eubacterial and archaeal structure-specific 5'-exonucleases.
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The 5'-exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN1 proteins of Eukarya and Archaea are members of a family of structure-specific 5'-exonucleases with similar function but limited sequence similarity. Their physiological role is to remove the displaced 5' strands created by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase. In this paper, we define the substrate requirements for the 5'-exonuclease enzymes from Thermus aquaticus, Thermus thermophilus, Archaeoglobus fulgidus, Pyrococcus furiosus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum. The optimal substrate of these enzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex that overlaps the downstream duplex by one base pair. That single base of overlap causes the enzymes to leave a nick after cleavage and to cleave several orders of magnitude faster than a substrate that lacks overlap. The downstream duplex needs to be 10 base pairs long or greater for most of the enzymes to cut efficiently. The upstream duplex needs to be only 2 or 3 base pairs long for most enzymes, and there appears to be interaction with the last base of the primer strand. Overall, the enzymes display very similar substrate specificities, despite their limited level of sequence similarity. (+info)
The reaction mechanism of ribonuclease II and its interaction with nucleic acid secondary structures.
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Ribonuclease II is a processive 3'- to 5'-exoribonuclease in Escherichia coli with two binding sites: a catalytic site associated with the first few 3'-nucleotides and an anchor site binding nucleotides approximately 15 to 25 from the 3'-end. When RNase II degrades single-stranded helical poly(C), the enzyme-substrate complex dissociates at discrete intervals of 12 nucleotides. RNase II stalled at the last rC of single-stranded 3'-(rC)(n)(dC)(m) oligonucleotides. The more residues released, the faster the stalled complex dissociated and the less it inhibited RNase II activity, i.e. the enzyme-substrate association weakened progressively. Using phosphodiesterase I (PDE I) as a probe, a method was developed to identify cytidine residues in (32)P-oligonucleotides interacting with a protein. PAGE bands corresponding to nucleotides 1-6 from the 3'-end were consistent with interaction at the catalytic site, and following a gap, bands approximately 15 to 25 from the 3'-end, with anchor site association. Both 3' and 5' binding were necessary to maintain the complex. Of most significance, the original anchor site nucleotides remained fixed at the anchor site while the 3'-end was pulled, or threaded, through the catalytic site, i.e. the substrate did not 'slide' through the enzyme. DNA oligonucleotides with double-stranded stem-loops were good competitive inhibitors of RNase II. A 3'-single-stranded arm was essential, while optimal binding required both 5'- and 3'-arms. PDE I probing indicated that the nucleotides at the anchor site were specified by the spatial distance from the catalytic site, and on only one of the duplex strands. When degradation of a structured RNA paused or stopped, the RNase II-product commenced cycles of dissociation-reassociation. Duplex strand binding by RNase II made complex DNA or RNA structures accessible to degradation by other nucleases and further verified the PDE I footprinting method. (+info)
Developmental expression analysis of murine autotaxin (ATX).
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The murine homologue of the human motility-stimulating protein autotaxin (ATX) was identified as a BMP2 upregulated gene by subtractive cloning from mesenchymal progenitors C3H10T1/2 (Bachner, D., Ahrens, M., Betat, N., Schroder, D., Hoffmann. A., Lauber, J., Steinert, P., Flohe, L., Gross, G., 1998. Bmp-2 downstream targets in mesenchymal development identified by subtractive cloning from recombinant mesenchymal progenitors (C3H10T1/2). Dev. Dyn. 213, 398-411). ATX mRNA transcription is induced during BMP2 mediated osteo-/chondrogenic differentiation in vitro several orders of magnitude. To delineate a potential role for ATX in osteo-/chondrogenic development, its expression pattern during murine embryogenesis was examined in comparison with Col1a1 and Col2a1, a marker either of osteoblast, odontoblast and tendon or of chondrocyte development, respectively. Localization of murine ATX was first observed in the floor plate of the neural tube at day 9.5 of mouse embryonic development. Later, enhanced ATX expression levels were observed in proliferating subepithelial mesenchyme, during osteo-/chondrogenic and tooth development, in choroid plexus epithelium, in late kidney development, and in smooth muscles of the ductus deferens and the bladder. (+info)
Mass spectral characterization of a protein-nucleic acid photocrosslink.
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A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155. (+info)
Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells.
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Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, was originally isolated as a potent stimulator of tumor cell motility. In order to study whether ATX expression affects motility-dependent processes such as invasion and metastasis, we stably transfected full-length ATX cDNA into two non-expressing cell lines, parental and ras-transformed NIH3T3 (clone7) cells. The effect of ATX secretion on in vitro cell motility was variable. The ras-transformed, ATX-secreting subclones had enhanced motility to ATX as chemoattractant, but there was little difference in the motility responses of NIH3T3 cells transfected with atx, an inactive mutant gene, or empty vector. In MatrigelTM invasion assays, all subclones, which secreted enzymatically active ATX, demonstrated greater spontaneous and ATX-stimulated invasion than appropriate controls. This difference in invasiveness was not caused by differences in gelatinase production, which was constant within each group of transfectants. In vivo studies with athymic nude mice demonstrated that injection of atx-transfected NIH3T3 cells resulted in a weak tumorigenic capacity with few experimental metastases. Combination of ATX expression with ras transformation produced cells with greatly amplified tumorigenesis and metastatic potential compared to ras-transformed controls. Thus, ATX appears to augment cellular characteristics necessary for tumor aggressiveness. (+info)
Tritium sequence analysis of oligoribonucleotides: a combination of post-labeling and thin-layer chromatographic techniques for the analysis of partial snake venom phosphodiesterase digests.
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A tritium derivative method for sequence analysis of polyribonucleotides is detailed, which is based on borotritide reduction of oligonucleotide-3' dialdehydes generated by controlled snake venom phosphodiesterase/alkaline phosphomonoesterase digestion and periodate treatment of time point aliquots of the incubation mixture. Radioactive oligonucleotide derivatives are resolved according to chain length by PEI-cellulose(1) anion-exchange TLC and their 3'-termini identified by techniques described in the preceding paper of this series(2). The present tritium derivative method is compared with the one described previously(2). (+info)
Mode of action and application of Scorpion primers to mutation detection.
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Scorpion primers can be used to detect PCR products in homogeneous solution. Their structure promotes a unimolecular probing mechanism. We compare their performance with that of the same probe sequence forced to act in a bimolecular manner. The data suggest that Scorpions indeed probe by a unimolecular mechanism which is faster and more efficient than the bimolecular mechanism. This mechanism is not dependent on enzymatic cleavage of the probe. A direct comparison between Scorpions, TaqMan and Molecular Beacons on a Roche LightCycler indicates that Scorpions perform better, particularly under fast cycling conditions. Development of a cystic fibrosis mutation detection assay shows that Scorpion primers are selective enough to detect single base mutations and give good sensitivity in all cases. Simultaneous detection of both normal and mutant alleles in a single reaction is possible by combining two Scorpions in a multiplex reaction. Such favourable properties of Scorpion primers should make the technology ideal in numerous applications. (+info)
Synthesis and monitored selection of 5'-nucleobase-capped oligodeoxyribonucleotides.
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Oligodeoxynucleotides bearing 5'-appendages consisting of a nucleobase and an amide linkage were prepared from 5'-amino-5'-deoxyoligonucleotides, amino acid building blocks and thymine or uracil derivatives. Small chemical libraries of 5'-modified oligonucleotides bearing the nucleobase moieties via five, three or two atom linkages were subjected to spectrometrically monitored nuclease selections to identify members with high affinity for target strands. The smallest of the appendages tested, a uracil acetic acid substituent, was found to convey the greatest duplex stabilizing effect on the octamer 5'-T*GGTTGAC-3', where T* denotes the 5'-amino-5'-deoxythymidine residue. Compared to 5'-TTGGTTGAC-3', the modified sequence 5'-u-T*GGTTGAC-3' gives a duplex with 5'-GTCAACCAA-3' that melts 4 degrees C higher. The duplex-stabilizing effect of this 5'-substituent does not require a specific residue at the 3'-terminus of the complement and the available data suggest that the uracil moiety is located in the major groove of the duplex. (+info)