Abstract
The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined. Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA. The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization. In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities. This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate. However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e. the effects of the next nucleotide or the addition of deoxynucleoside monophosphates. These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate. This concept is discussed in relation to models for proofreading described in the literature.
Highlights
The implication is that the mutagenic effects of pyrophosphate allow one to probe the polymerase’s mechanism for increasing the fidelity of DNA synthesis
The most direct mechanism for increased frequency of misincorporation by pyrophosphate would bethe stimulation of selective pyrophosphorolysis of the last inserted complementary nucleotide immediately after formation of the phosphodiester bond [12]. This would be the opposite of conventional proofreading, in that the complementary nucleotide would be that one which is preferentially excised
Four observations argue against this mechanism: 1)AMV DNA polymerase [21] and DNA polymerase-a [26], like pol I, catalyze pyrophosphorolysis, yet the fidelity of these enzymes is not diminished by added pyrophosphate [24], even under conditionsin which synthesis is markedly diminished
Summary
Vol 261, No., Ieaue of October 15,pp. 13610-13616,1986 Printed in U.S.A. From The Joseph GottsteinMemorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington98195 and $TheNational Instituteof Environmental HealthSciences, Research TrianglePark, North Carolina 27709. The pyrophosphate-induceidnfidelity Kornberg with pol I’ (Z), from genetic and biochemical data has a differentspecificity from, andis not competitive with phage T4-induced DNA polymerase [9,10,11], and more with, two experimental markers 3o’f4 5’ exonuclease recently from experiments which directly measure the mutaproofreading; i.e. the effects of the nucleotide or genic consequences of decreased exonuclease activity during thaedditioondfeoxynucleosidme onophosphates These distinctive features suggest a second modeof is proposed is referred to askinetic proofreading. Resynthesis was carried out in a 5-ml reaction mixture containing 1.14 mg of exonuclease 111-treatedDNA; 50 mM Tris-HC1 (pH 7.4); 2 m M MgCl,; 50 p~ each dCTP,dTTP,and dGTP; 98 p M [3Cs]dGTP (40,000 dpm/pmol); and 95 pg of homogeneous E. coli DNA polymerase I. Within experiments, reversion frequencies of duplicates usually varied by less than 20%
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