O4-Methyl-, O4-ethyl-, and O4-isopropylthymidine 5'-triphosphates as analogues of thymidine 5'-triphosphate: kinetics of incorporation by Escherichia coli DNA polymerase I.

Biochemistry (1986), Volume 25, Page 1201

Abstract:

O4-Methyl-, O4-ethyl-, and O4-isopropylthymidine 5'-triphosphates, which can be formed by N-nitroso carcinogens, were tested for their ability to substitute for thymidine 5'-triphosphate (dTTP) in synthesis catalyzed by Escherichia coli DNA polymerase I (Pol I) by using activated DNA or synthetic polymers as templates. All could substitute for dTTP for short periods, the rate and extent decreasing with the size of the alkyl group. Because the structure of O4-alkylthymidine does not permit normal hydrogen bond formation with deoxyadenosine, it was inferred that eventual formation of a poor or frayed primer end was responsible for termination of synthesis. Synthesis of polymers at temperatures ranging from 0 to 40 degrees C showed that the extent of incorporation using the O4-alkyl-dTTPs was favored, relative to dTTP, when the terminal helical structure was stabilized by low temperatures. Kmapp values were determined for each O4-alkyldeoxynucleoside 5'-triphosphate. These values were 0.7 microM for dTTP, 5 microM for methyl-dTTP, 11 microM for ethyl-dTTP, and 33 microM for isopropyl-dTTP. O4-Alkyl-dTTPs were tested for their ability to inhibit or compete with dTTP incorporation and found to have a minimal effect, even when present at high concentration. These experiments indicated that Pol I can incorporate deoxynucleotides with O4-alkyl substituents into an ordered DNA structure. A postulated base-pairing scheme with deoxyadenosine is described.

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