5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA.

Abstract

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 .mu.M 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 labeling reagents.