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

Journal Article · Tue Feb 10 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00377a042· OSTI ID:6528681

Ide, H; Melamede, R J; Wallace, S S

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 Ag/sub 2/O. 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 (/sup 3/H)DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The result suggests that Escherichia coli DNA polymerase I uses both isomers of DHdTTP as substrates and that the overall efficiency of incorporation is primarily determined by the concentration of the isomers in the nucleotide pool.

• Research Organization: New York Medical College, Valhalla
• DOE Contract Number: AC02-80EV10417
• OSTI ID: 6528681
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:3; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE PHOSPHATASE
BACTERIA
BIOCHEMISTRY
BIOSYNTHESIS
CHEMISTRY
CHROMATOGRAPHY
DNA POLYMERASES
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
ESTERASES
HYDROLASES
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
MICROORGANISMS
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHATASES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
SEPARATION PROCESSES
SUBSTRATES
SYNTHESIS
THYMIDYLIC ACID
TRANSFERASES
TRITIUM COMPOUNDS