Reznikoff William S.

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Reznikoff
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William S.
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  • Article
    Tn5 synaptic complex formation : role of transposase residue W450
    (American Society for Microbiology, 2007-12-14) Gradman, Richard J. ; Reznikoff, William S.
    A series of Tn5 transposases (Tnp's) with mutations at the conserved amino acid position W450, which was structurally predicted to be important for synapsis, have been generated and characterized. This study demonstrates that W450 is involved in hydrophobic (and possibly aromatic) contacts within the Tnp monomer that negatively regulate synaptic complex formation.
  • Article
    Comparative sequence analysis of IS50/Tn5 transposase
    (American Society for Microbiology, 2004-12) Reznikoff, William S. ; Bordenstein, Seth R. ; Apodaca, Jennifer
    Comparative sequence analysis of IS50 transposase-related protein sequences in conjunction with known structural, biochemical, and genetic data was used to determine domains and residues that play key roles in IS50 transposase function. BLAST and ClustalW analyses have been used to find and analyze six complete protein sequences that are related to the IS50 transposase. The protein sequence identity of these six homologs ranged from 25 to 55% in comparison to the IS50 transposase. Homologous motifs were found associated with each of the three catalytic residues. Residues that play roles in transposase-DNA binding, protein autoregulation, and DNA hairpin formation were also found to be conserved in addition to other residues of unknown function. On the other hand, some homologous sequences did not appear to be competent to encode the inhibitor regulatory protein. The results were also used to compare the IS50 transposase with the more distantly related transposase encoded by IS10.
  • Article
    A bifunctional DNA binding region in Tn5 transposase
    (Blackwell, 2007-12-16) Gradman, Richard J. ; Ptacin, Jerod L. ; Bhasin, Archna ; Reznikoff, William S. ; Goryshin, Igor Y.
    Tn5 transposition is a complicated process that requires the formation of a highly ordered protein–DNA structure, a synaptic complex, to catalyse the movement of a sequence of DNA (transposon) into a target DNA. Much is known about the structure of the synaptic complex and the positioning of protein–DNA contacts, although many protein–DNA contacts remain largely unstudied. In particular, there is little evidence for the positioning of donor DNA and target DNA. In this communication, we describe the isolation and analysis of mutant transposases that have, for the first time, provided genetic and biochemical evidence for the stage-specific positioning of both donor and target DNAs within the synaptic complex. Furthermore, we have provided evidence that some of the amino acids that contact donor DNA also contact target DNA, and therefore suggest that these amino acids help define a bifunctional DNA binding region responsible for these two transposase–DNA binding events.
  • Article
    Site-directed mutagenesis studies of Tn5 transposase residues involved in synaptic complex formation
    (American Society for Microbiology, 2007-08-10) Vaezeslami, Soheila ; Sterling, Rachel ; Reznikoff, William S.
    Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.
  • Article
    A novel conjugative transposon carrying an autonomously amplified plasmid
    (American Society for Microbiology, 2024-01-23) Vineis, Joseph H. ; Reznikoff, William S. ; Antonopoulos, Dionysios A. ; Koval, Jason ; Chang, Eugene B. ; Fallon, Bailey R. ; Paul, Blair G. ; Morrison, Hilary G. ; Sogin, Mitchell L.
    Tetracyclines serve as broad-spectrum antibiotics to treat bacterial infections. The discovery of new tetracycline resistance genes has led to new questions about the underlying mechanisms of resistance, gene transfer, and their relevance to human health. We tracked changes in the abundance of a 55-kbp conjugative transposon (CTn214) carrying tetQ, a tetracycline resistance gene, within a Bacteroides fragilis metagenome-assembled genome derived from shotgun sequencing of microbial DNA extracted from the ileal pouch of a patient with ulcerative colitis. The mapping of metagenomic reads to CTn214 revealed the multi-copy nature of a 17,044-nt region containing tetQ in samples collected during inflammation and uninflamed visits. B. fragilis cultivars isolated from the same patient during periods of inflammation harbored CTn214 integrated into the chromosome or both a circular, multi-copy, extrachromosomal region of the CTn214 containing tetQ and the corresponding integrated form. The tetracycline-dependent mechanism for the transmission of CTn214 is nearly identical to a common conjugative transposon found in the genome of B. fragilis (CTnDOT), but the autonomously amplified nature of a circular 17,044-nt region of CTn214 that codes for tetQ and the integration of the same sequence in the linear chromosome within the same cell is a novel observation. Genome and transcriptome sequencing of B. fragilis cultivars grown under different concentrations of tetracycline and ciprofloxacin indicates that tetQ in strains containing the circular form remains actively expressed regardless of treatment, while the expression of tetQ in strains containing the linear form increases only in the presence of tetracycline.