Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

Abstract

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)

Human DNA Polymerase κ Bypasses and Extends beyond Thymine Glycols during Translesion Synthesis in Vitro, Preferentially Incorporating Correct Nucleotides

“…Thymine Glycol Template 1 (TgBr 2 ) Has a Lower Proportion of 5R Stereoisomers)-5,6-Dihydro-5,6-dihydroxythymidine-5Ј-triphosphate (thymidine glycol-5Ј-triphosphate) was synthesized according to a previously published protocol (34) and characterized by HPLC (one peak), 31 P NMR (three lines), 1 H NMR, and ESI mass spectrometry ((M-H ϩ ) 1Ϫ ϭ 515 atomic mass units). Additionally, the stereochemical composition of this sample was assayed by digestion of the deoxynucleoside triphosphate with alkaline phosphatase and separation of the deoxynucleosides by HPLC according to a previously published method (33).…”

Enzymatic Processing of Uracil Glycol, a Major Oxidative Product of DNA Cytosine

“…Chemicals, Enzymes, and DNA-dTgTP and dUgTP were prepared as described previously (24,31); the 2Ј-deoxynucleoside triphosphates used in the DNA polymerase reactions and the Mono-Q 5/5 column were purchased from Pharmacia; Partisphere SAX, 0.4 ϫ 12.5 cm, column was obtained from Whatman; 2Ј,3Ј-dideoxynucleoside triphosphates, Klenow fragment (10 M/l), Sequenase version 2.0 (13 M/l), T4 DNA ligase (5 M/l), and shrimp alkaline phosphatase were obtained from U. S. Biochemical Corp.; terminal transferase (10 M/ml) and T4 polynucleotide kinase were purchased from Boehringer Mannheim; uracil DNA glycosylase (100 M/l) was obtained from Epicentre Technologies; E. coli endo III (10 M), endo VIII (200 M), and Fpg DNA glycosylase (6.6 M) were purified as described previously (16).…”

“…We have previously reported the synthesis of dUgTP and shown it to be a reasonably efficient substrate for DNA polymerase I Kf (31). This is in contrast to its structural relative, dTgTP, which is a poor substrate (24,31). Both dUgTP and dTgTP are incorporated in place of T (31) and thus would not be potentially mutagenic if incorporated from oxidized nucleotide pools.…”

Targeting One-Carbon Metabolism in Breast Cancer

“…We suggest that this EMT-dependent metabolic rewiring, which activates only selected components of a given metabolic pathway, is not exclusive to DPYD, Ide, 1988;Ide et al, 1987). It remains to be determined whether such modified nucleotides can be produced in human cells and, if so, how they affect cellular phenotypes.…”