Studies on the mechanism of Escherichia coli DNA polymerase I large fragment. Chain termination and modulation by polynucleotides.

Abstract:

Homopolymer replication systems and measurement of precise product chain length have been used to elucidate two new points about the mechanism of Escherichia coli DNA polymerase I large fragment: chain termination as a function of product chain length is multiphasic, and polynucleotides exert a secondary effect in the mechanism of this enzyme. During replication of (dT)800 or (dA)800 with short oligonucleotides as primer, DNA polymerase I large fragment was processive, catalyzing hundreds of dNMP incorporations during each cycle of binding to the template-primer, incorporation, and termination. Our observations indicated, however, that polynucleotides could terminate chain elongation and that this effect probably occurred through interaction at a secondary binding site on the enzyme. Thus, in the presence of higher levels of template-primer, early termination occurred and the relatively short product molecules could be resolved by gel electrophoresis. Incubation conditions were adjusted so that the number of product molecules at each chain length was equal to the actual number of termination events, and, therefore, the statistical chance for termination as a function of product chain length could be calculated. These termination probability values depended upon specific incubation conditions, such as dNTP level and whether the primer was a ribo- or deoxyribonucleotide, and interestingly, the values changed as the chain length of the product increased. For the first 5 to 10 dMP residues added to the primer, termination probability declined with each dNMP addition, but then remained constant for the addition of the next 20 to 40 dNMP residues. These results are discussed in the context of a kinetic model representing two stages of synthesis during the formation of each product molecule.

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