5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA.
We have synthesized the photoactive deoxyuridine nucleotide 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP) and used it to synthesize light-sensitive DNA by enzymatic incorporation. In the absence of ultraviolet light, 5-N3dUTP is a substrate for Escherichia coli DNA polymerase I. In in vitro DNA synthesis reactions using bacteriophage M13 single-stranded DNA as the template and 5-N3dUTP in place of dTTP, a photoactive complementary strand was synthesized by DNA polymerase I. The complementary strand was not synthesized when the 5-N3dUTP was substituted for dCTP or when it was exposed to ultraviolet light prior to the addition of DNA polymerase I. Using a synthetic lac operator template of 26 bases and a 15-base primer, we generated a photoactive 26-base-pair lac operator by enzymatically incorporating 5-N3dUMP with DNA polymerase I. Crosslinking of this photoactive DNA fragment to lac repressor was totally dependent on the presence of UV light and was reduced 78% by 150 microM isopropyl beta-D-thiogalactoside. Under the same conditions no crosslinking to lac repressor was observed using a nonphotoactive 26-base-pair lac operator. Photoactivatable deoxyuridine analogs have potential application as reagents to crosslink DNA binding proteins to 5-azidouracil-containing DNA and as active-site-directed photoaffinity labelling reagents.
Evans
R K
RK
Johnson
J D
JD
Haley
B E
BE
D000345:Affinity Labels; D001386:Azides / Q000528:radiation effects; D003432:Cross-Linking Reagents; D004247:DNA / Q000528:radiation effects; D004256:DNA Polymerase I / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000528:radiation effects; D003856:Deoxyuracil Nucleotides / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D010777:Photochemistry; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D003856:Deoxyuracil Nucleotides; C048865:5-azido-2'-deoxyuridine 5'-triphosphate; D004247:DNA; D004256:DNA Polymerase I