Synthesis and biological properties of 5-azido-2'-deoxyuridine-5'-triphosphate: a photoaffinity labelling reagent and a tool for the enzymatic synthesis of photoactive DNA
To produce light sensitive DNA for the study of protein-DNA interactions, 5-azido-2'-deoxyuridine-5'-triphosphate (5-N/sub 3/dUTP), was synthesized to serve as a substrate for the enzymatic incorporation of a photoactive nucleotide analog into DNA. 5-N-dUTP was synthesized from dUMP in five steps with the key reaction being the nitration of dUMP to 5-NO/sub 2/dUMP in 98% yield using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. The triphosphate form of the nucleotide (5-N/sub 3/dUTP), formed through a chemical coupling of pyrophosphate to 5-N/sub 3/dUMP, was found to support DNA synthesis by E. coli DNA polymerase I at a rate indistinguishable to that supported by dTTP. Using UMP as the starting compound 5-N/sub 3/UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare (..gamma../sup 32/P) 5-N/sub 3/dUTP for use as an active site directed photoaffinity labeling reagent, a simple method of preparing (..gamma../sup 32/P) labeled pyrimidine nucleotides was developed. (..gamma../sup 32/P) 5-N/sub 3/dUTP is an effective photoaffinity labeling reagent for DNA polymerase I which binds to the active site of the enzyme with a two fold higher affinity than dTTP.
- Research Organization:
- Wyoming Univ., Laramie (USA)
- OSTI ID:
- 5348975
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
Similar Records
(5'-/sup 32/P)-8-azidoguanosine-3',5'-monophosphate. I. Synthesis and properties. II. Interaction with E. coli proteins
Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins
Related Subjects
DNA
BIOSYNTHESIS
UTP
CHEMICAL PREPARATION
CHEMICAL PROPERTIES
DNA POLYMERASES
ESCHERICHIA COLI
PHOSPHORUS 32
REAGENTS
TRACER TECHNIQUES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
SYNTHESIS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques