Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have evaluated a genotoxicity assay that combines in situ end-labeling, colloidal gold tagging and electron microscopy in order to adapt it to the measurement of in vitro biomaterial-induced genotoxicity. Human lymphocytes were cultured in semi-physiological medium which had been previously exposed to biomaterial extracts of commercially pure titanium following ISO standards. In order to visualize the location of induced DNA strand breaks, cells were then exposed to exonuclease III which partially digests and amplifies lesions by releasing nucleotides at free 3' hydroxyl ends from nicked double-stranded DNA. The resulting single-stranded DNA was allowed to hybridize with short oligonucleotides of random sequences including biotinylated dUTP. After random priming using Escherichia coli DNA polymerase I, incorporation of biotin-dUTP was detected by immunogold binding to the chromatin. Cells exposed to a mutagenic concentration of methyl methanesulfonate, as a positive control, showed a significantly higher and stronger gold staining than both titanium-exposed and unexposed specimens. This assay allows a precise localization and quantification of both in vitro DNA breakage and DNA repair. It could provide a powerful tool for rapid assessment of the genotoxic potential of new biomaterials.
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PMID:Immunogold electron microscopy in situ end-labeling (EM-ISEL): assay for biomaterial DNA damage detection. 962 7

We developed a titanium-binding-peptide-1 (TBP-1)-tagged DNA polymerase, for self-oriented immobilization onto a titanium oxide (TiO2) substrate. The enzymatic function of a polymerase immobilized on a solid state device is strongly dependent on the orientation of the enzyme. The TBP-tagged DNA polymerase, which was derived from a hyperthermophilic archaeon, was designed to incorporate the RKLPDA peptide at the N-terminus, and synthesized by translation processes in Escherichia coli (E. coli). The specific binding of the TBP-tagged DNA polymerase onto a TiO2 substrate was clearly monitored by surface plasmon resonance spectroscopy (SPR) and by surface potential detection with an extended-gate field effect transistor (FET). In the SPR analyses, constant quantities of the DNA polymerase were stably immobilized on the titanium substrate under flow conditions, regardless of the concentration of the DNA polymerase, and could be completely removed by a 4 M MgCl2 wash after measurement. The FET signal showed the contribution of the molecular charge in the TBP motif to the binding with TiO2. In addition, the TBP-tagged DNA polymerase-tethered TiO2 gate electrode enabled the effective detection of the positive charges of hydrogen ions produced by the DNA extension reaction, according to the FET principle. Therefore, the self-oriented immobilization platform based on the motif-inserted enzyme is suitable for the quick and stable immobilization of functional enzymes on biosensing devices.
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PMID:Self-oriented immobilization of DNA polymerase tagged by titanium-binding peptide motif. 2551 38