DNA synthesis in vitro : proceedings of the Second Annual Harry Steenbock Symposium held in Madison, Wisconsin on July 10-12, 1972 / edited by R.D. Wells and R.B. Inman.

  • Harry Steenbock Symposium
Date:
©1973, [1974]
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Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: DNA synthesis in vitro : proceedings of the Second Annual Harry Steenbock Symposium held in Madison, Wisconsin on July 10-12, 1972 / edited by R.D. Wells and R.B. Inman. Source: Wellcome Collection.

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    52 NOSSAL AND HERSHFIELD 0.2 Free dAMP ß 0 30 MINUTES \/ / 60 Fig. 5. Template hydrolysis and dATP uti¬ lization for Escherichia coli DNA treated with exonuclease III. The standard reaction mixture contained ['С] E. co/i DNA (2.4 X 10''срт/м.то1е of nucleotide phosphate) degraded to 25% acid solubility with exonu¬ clease III, 0.1 mM; dTTP, dCTP, dGTP, and ['H]dATP (4.4 x 10^ cpm/pimole), each at 0.1 mM; and T4 DNA polymerase, 3.15 jJLg/ml. Incubation was at 37°С, and 10-jil samples were removed for analysis at the times indicated. Reproduced from reference 13. defined sequence, that only unpaired nucleotides at the 3' chain terminus are removed before the chain is extended (2). Hydrolysis during Synthesis In contrast to the limited hydrolysis of denatured DNA before synthesis begins, we have found that there is considerable hydrolysis of newly incorporated nucleotides. This is conveniently measured by the DNA- dependent conversion of deoxynucleoside triphosphates to free deoxynu- cleoside monophosphates (Reactions 1 and 2, Fig. 3, and Table 2) (13). The table shows that complementary homopolymers which can serve as template and primer are required for the conversion of each of the four dNTPs to its free dNMP. The individual homopolymers are not sufficient. In addition, a free 3'-hydroxyl group on the primer strand and a chain length of more than seven are required. The B22 protein, which cannot support incorporation, does not catalyze this conversion of dNTP to dNMP. The large extent to which this hydrolysis of newly formed DNA occurs during synthesis in vitro is shown in Fig. 5. The incorporation of dAMP from dATP into DNA is approximately that expected for complete repair of the DNA removed by exonuclease III. The formation of free dAMP from dATP occurs at about 40% of the initial rate of stable incorporation, and continues at this pace until the supply of tri¬ phosphates is depleted. The rates of stable dNMP incorporation and con¬ version of dNTP to dNMP depend in a similar way on the concentrations of template, triphosphates, and enzyme (13). However, while stable in¬ corporation requires all four triphosphates, considerable incorporation and subsequent hydrolysis do occur when less than a full complement is present.