Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. II. Phosphorylation by phage T4 polynucleotide kinase
Phage T4 polynucleotide kinase proved incapable of catalyzing the phosphorylation of thymidylyl-(3'----5')-thymidine containing either a cis-syn-cyclobutane pyrimidine dimer (d-T less than p greater than T) or a 6-4'-(pyrimidin-2'-one)pyrimidine photoproduct (d-T(p)-T), and similarly the UV-modified compounds of (dT)3 bearing either photoproduct at their 5'-end (d-T less than p greater than TpT and d-T(p)TpT). In contrast, the 3'-structural isomers of these trinucleotides (d-TpT less than p greater than T and d-TpT(p)T) were phosphorylated at the same rate as the parent compound. These phosphorylatable lesion-containing oligonucleotides are quantitatively released from UV-irradiated poly(dA):poly(dT) by enzymatic hydrolysis with snake venom phosphodiesterase and alkaline phosphatase. By combining this digestion regimen with phosphorylation by polynucleotide kinase and (gamma-/sup 32/P)ATP, pyrimidine dimers were quantitated at the fmol level following exposure of poly(dA):poly(dT) and herring sperm DNA to biologically relevant UV fluences. The rate of dimer induction in the synthetic polymer, approximately 10 dimers/10(6) nucleotides/Jm-2, was in close agreement with that obtained by conventional methods. Dimers were induced at one-fourth of this rate in the natural DNA. Further treatment of the phosphorylated oligonucleotides derived from irradiated herring sperm DNA with nuclease P1 released the labeled 5'-nucleotide, thus permitting analysis of the nearest-neighbor bases 5' to the lesions. We observed a ratio for pyrimidine-to-purine bases of almost 6:1, implicating tripyrimidine stretches as hotspots for UV-induced DNA damage.
- Research Organization:
- Cross Cancer Institute, Edmonton, Alberta (Canada)
- OSTI ID:
- 6024203
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 264:11
- Country of Publication:
- United States
- Language:
- English
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NUCLEOTIDES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
ENZYME ACTIVITY
PYRIMIDINE DIMERS
RADIOINDUCTION
ATP
BACTERIOPHAGES
CHEMICAL RADIATION EFFECTS
DOSE-RESPONSE RELATIONSHIPS
FISHES
IN VITRO
NUCLEOTIDASES
OLIGONUCLEOTIDES
PHOSPHORUS 32
SPERMATOZOA
THYMIDINE
THYMINE
TRACER TECHNIQUES
ULTRAVIOLET RADIATION
ANIMALS
AQUATIC ORGANISMS
AZINES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
ELECTROMAGNETIC RADIATION
ENZYMES
ESTERASES
GAMETES
GERM CELLS
HETEROCYCLIC COMPOUNDS
HYDROLASES
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOSIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PARASITES
PHOSPHATASES
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PYRIMIDINES
RADIATION CHEMISTRY
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
RIBOSIDES
TRANSFERASES
URACILS
VERTEBRATES
VIRUSES
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture