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
Thesis/Dissertation
·
OSTI ID:5348975
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
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOSYNTHESIS
CHEMICAL PREPARATION
CHEMICAL PROPERTIES
DAYS LIVING RADIOISOTOPES
DNA
DNA POLYMERASES
ENZYMES
ESCHERICHIA COLI
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
REAGENTS
SYNTHESIS
TRACER TECHNIQUES
TRANSFERASES
UTP
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOSYNTHESIS
CHEMICAL PREPARATION
CHEMICAL PROPERTIES
DAYS LIVING RADIOISOTOPES
DNA
DNA POLYMERASES
ENZYMES
ESCHERICHIA COLI
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
REAGENTS
SYNTHESIS
TRACER TECHNIQUES
TRANSFERASES
UTP