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From the
1 From the Laboratory of Biochemical Pharmacology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014
We have studied the 3' to 5' exonucleolytic activity of the T4 DNA polymerase during in vitro DNA synthesis. By separating reaction products by one-dimensional thin layer chromatography we were able to follow simultaneously nucleotide incorporation, hydrolysis of newly incorporated nucleotides, and also the degradation of thymine-labeled template DNA. When all four deoxynucleoside triphosphates are present, degradation of template DNA appears to be limited to the removal of unpaired 3'-terminal regions early in the reaction; addition of nucleotides to paired termini protects template molecules from exonucleolytic attack. When one or more deoxynucleoside triphosphates are omitted, the extent of template degradation depends upon the relative rates of hydrolysis and incorporation of the available substrates. During copying of single stranded DNA, hydrolysis of newly incorporated nucleotides, as shown by the appearance of free deoxynucleoside monophosphates derived from all four triphosphates, occurs at a rate slower than, but related to, the initial rate of synthesis. Free deoxynucleoside monophosphate formation continues after product DNA has ceased to accumulate. At 37° free dAMP is formed at 20 to 40% of the initial rate of dAMP incorporation. During a reaction in which the four deoxynucleotides are incorporated into product DNA at similar rates and to about the same extent, newly added dAMP is hydrolyzed 1 As a possible means of studying the frequency of errors in copying, we have measured the incorporation and the hydrolysis of residues derived from deoxynucleoside triphosphates that are not complementary to either strand of homopolymer duplexes. With poly(dA)·poly(dT) as template-primer, both dGTP and dCTP were converted to free dGMP and dCMP, although neither appeared in product polymer. Thus misincorporation had occurred, but the nucleotides were subsequently removed by the exonuclease activity of the polymerase.
Hydrolysis of Template and Newly Synthesized Deoxyribonucleic Acid by the 3' to 5' Exonuclease Activity of the T4 Deoxyribonucleic Acid Polymerase
to 3 times as often as dTMP and dGMP, and 6 to 9 times more frequently than dCMP, suggesting that the polymerase recognizes specific bases. At 20° the rate of hydrolysis of newly incorporated nucleotides is depressed to a greater degree than the rate of synthesis, suggesting that local unwinding of the 3' terminus is required for hydrolysis of newly incorporated residues. The T4 polymerase has previously been found by Englund to carry out repeated hydrolysis and replacement of nucleotides at the ends of native DNA without net synthesis or degradation. We find that triphosphate utilization (net incorporation plus hydrolysis of newly incorporated residues) is faster with native than denatured T7 DNA. However, when both forms of T7 DNA are present in a reaction, incorporation and release of deoxynucleotides occur at the rate seen with denatured DNA alone, even when enzyme is in molar excess of total 3'-hydroxyl chain termini.
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