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Title: 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 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.more » 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.« less

Authors:
; ;
Publication Date:
Research Org.:
New York Medical College, Valhalla
OSTI Identifier:
6528681
DOE Contract Number:  
AC02-80EV10417
Resource Type:
Journal Article
Journal Name:
Biochemistry; (United States)
Additional Journal Information:
Journal Volume: 26:3
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ALKALINE PHOSPHATASE; BIOCHEMISTRY; NUCLEOTIDES; BIOSYNTHESIS; TRITIUM COMPOUNDS; DNA POLYMERASES; ENZYME ACTIVITY; ESCHERICHIA COLI; LIQUID COLUMN CHROMATOGRAPHY; SUBSTRATES; THYMIDYLIC ACID; BACTERIA; CHEMISTRY; CHROMATOGRAPHY; ENZYMES; ESTERASES; HYDROLASES; LABELLED COMPOUNDS; MICROORGANISMS; NUCLEOTIDYLTRANSFERASES; ORGANIC COMPOUNDS; PHOSPHATASES; PHOSPHORUS-GROUP TRANSFERASES; POLYMERASES; SEPARATION PROCESSES; SYNTHESIS; TRANSFERASES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Ide, H, Melamede, R J, and Wallace, S S. Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I. United States: N. p., 1987. Web. doi:10.1021/bi00377a042.
Ide, H, Melamede, R J, & Wallace, S S. Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I. United States. https://doi.org/10.1021/bi00377a042
Ide, H, Melamede, R J, and Wallace, S S. 1987. "Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I". United States. https://doi.org/10.1021/bi00377a042.
@article{osti_6528681,
title = {Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I},
author = {Ide, H and Melamede, R J and Wallace, S S},
abstractNote = {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.},
doi = {10.1021/bi00377a042},
url = {https://www.osti.gov/biblio/6528681}, journal = {Biochemistry; (United States)},
number = ,
volume = 26:3,
place = {United States},
year = {Tue Feb 10 00:00:00 EST 1987},
month = {Tue Feb 10 00:00:00 EST 1987}
}