Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. II. Phosphorylation by phage T4 polynucleotide kinase.
Phage T4 polynucleotide kinase (EC 2.7.1.78) 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 (Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989) J. Biol. Chem. 264, 6355-6363). By combining this digestion regimen with phosphorylation by polynucleotide kinase and [gamma-32P]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.
Weinfeld
M
M
Department of Medicine, Cross Cancer Institute, Edmonton, Alberta, Canada.
Liuzzi
M
M
Paterson
M C
MC
D000818:Animals; D001482:Base Composition; D002851:Chromatography, High Pressure Liquid; D004307:Dose-Response Relationship, Radiation; D005399:Fishes; D066298:In Vitro Techniques; D008297:Male; D009708:Nucleotidases / Q000378:metabolism; D009841:Oligonucleotides / Q000032:analysis; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism*; D011740:Pyrimidine Dimers / Q000032:analysis* / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013094:Spermatozoa; D013938:Thymidine Monophosphate / Q000378:metabolism / Q000528:radiation effects*; D013942:Thymine Nucleotides / Q000528:radiation effects*; D014466:Ultraviolet Rays
D009841:Oligonucleotides; D011740:Pyrimidine Dimers; D013942:Thymine Nucleotides; D013938:Thymidine Monophosphate; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D009708:Nucleotidases; D015719:Single-Strand Specific DNA and RNA Endonucleases