Incorporation of biotin-labeled deoxyuridine triphosphate into DNA during excision repair and electron microscopic visualization of repair patches.
Biotin-labeled deoxyuridine triphosphate (BiodUTP) has the potential to be a useful affinity probe for studies on DNA repair, if it can be incorporated into DNA repair patches and does not inhibit subsequent steps in the excision repair pathway. We have synthesized BiodUTP by an improved procedure and have used permeable normal human fibroblasts to determine the effect of substituting BiodUTP for thymidine triphosphate on several steps in the excision repair pathway: incision, polymerization, ligation, and nucleosome rearrangement. The results demonstrate that BiodUTP is efficiently incorporated into repair patches and has little or no effect on the repair process. The presence of BiodUMP in ligated repair patches has been used to visualize the repair patches by electron microscopy following incubation with ferritin-labeled avidin. This approach has been used to estimate the maximum size of repair patches induced by ultraviolet radiation.
D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication / Q000528:radiation effects; D004269:DNA, Bacterial / Q000302:isolation & purification; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D008854:Microscopy, Electron; D009707:Nucleosomes / Q000648:ultrastructure; D011067:Poly dA-dT / Q000378:metabolism*; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D014466:Ultraviolet Rays
D004269:DNA, Bacterial; D003856:Deoxyuracil Nucleotides; D009707:Nucleosomes; D011089:Polydeoxyribonucleotides; D011067:Poly dA-dT; D001710:Biotin; D004256:DNA Polymerase I