We present a method for rapid measurement of DNA-protein interactions using voltage-driven threading of single DNA molecules through a protein nanopore. Electrical force applied to individual ssDNA-exonuclease I complexes pulls the two molecules apart, while ion current probes the dissociation rate of the complex. Nanopore force spectroscopy (NFS) reveals energy barriers affecting complex dissociation. This method can be applied to other nucleic acid-protein complexes, using protein or solid-state nanopore devices.
The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5′–3′ direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5′ single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg2+ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg2+ confirmed that substrates with 5′ tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading ∼1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5′ phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ.
Glycosylasparaginase is an N-terminal nucleophile hydrolase and is activated by intramolecular autoproteolytic processing. This cis-autoproteolysis possesses unique kinetics characterized by a reversible N-O acyl rearrangement step in the processing. Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved in binding of free amino acids in the partially formed substrate pocket on certain mutant precursors. This binding site is sequestered in the wild-type precursor. Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the individual mutants and amino acids. The polypeptide sequence at the processing site, which is highly conserved, adopts a special conformation. Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket through interaction with Arg-180. The reactive nucleophile Thr-152 is activated not only by deprotonation by His-150 but also by interaction with Thr-170, suggesting a His-Thr-Thr active triad for the autoproteolysis.
Initially, a cis-enediol mechanism was proposed for D-xylose isomerase (14,22), similar to the mechanism of triose-phosphate isomerase. However, isotope exchange experiments (23) and crystallographic analyses (15, 16) with various substrates and inhibitors suggests that the reaction proceeds via a metalmediated hydride shift (Fig. 1a). The currently accepted pathway for the reaction involves the preferential binding of ␣-Dxylopyranose (24, 25) followed by ring opening (25), extension of the substrate, and then the hydride shift (15,16,26). Recently, Meng et al. (27) have proposed that the hydride shift occurs on the cyclic form of sugar (Fig. 1b).Site-directed mutagenesis has been used to probe the functions of specific active site residues in D-xylose isomerase (27-32), however, only a few structures of mutant enzymes have been reported (19,(33)(34)(35). Kinetic data can be misleading if the substitutions affect the properties of catalytically important residues other than those changed by mutagenesis. For this reason, we have shifted our mutagenic studies from the Escherichia coli D-xylose isomerase (28, 29), which has not been successfully crystallized, to the S. rubiginosus enzyme which readily forms crystals diffracting x-rays beyond 2.0 Å (15,34,36 MATERIALS AND METHODSBacterial Strains and Plasmids-S. rubiginosus (ATCC 21175) was obtained from the American Type Culture Collection. E. coli BL21(DE3) (F Ϫ ompT r B Ϫm B Ϫ ( cTts857 ind1 Sam7 nin5 lacUV5-T7 gene 1)) was from Novagen. E. coli TG1 (supE hsd ⌬5 thi ⌬(lac-proAB) FЈ (traD36 proAB ϩ lacI q lacZ ⌬M15)) was used to propagate plasmid and bacteriophage M13. M13 mp18 and M13 mp19 DNA were purchased from U. S. Biochemical Corp. and pET11d plasmid DNA was from Novagen. S. rubiginosus xylA cloned into pET11d is called pRDW100 (its construction is described below) and is regulated by T7 RNA polymerase and LacZ, using the strain BL21(DE3) as a host.Biochemical Reagents-All compounds were reagent grade and purchased from Sigma, except acetaldehyde and 5-thio-␣-D-glucopyranose (THG), 1 which were from Aldrich. The sugar 5-deoxy-D-xylulose was synthesized by aldol condensation of dihydroxyacetone and acetaldehyde, using rabbit muscle aldolase with the cofactor sodium arsenate as a catalyst, and was purified by ion-exchange chromatography, 2 using a scheme similar to that described by Durrwachter et al. (37) for the synthesis of 5-deoxy-D-fructose.DNA Isolation, Transformation, and Manipulations-S. rubiginosus was grown in yeast and maltose extract medium supplemented with 34% (w/v) sucrose and chromosomal DNA was isolated as Hopwood et al. (38). Both plasmid and bacteriophage DNA were isolated from cul-
Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M. G. Johnson, Appi. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination ofL. nwnocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.
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