On the fidelity of DNA replication. Specificity of nucleotide substitution by intercalating agents.

C W Shearman,L A Loeb

doi:10.1016/s0021-9258(18)32648-6

Abstract

The effects of intercalating agents on the fidelity of DNA synthesis in vitro have been investigated. The accuracy of DNA synthesis with Escherichia coli DNA polymerase I with both the poly[d(A-T)] and poly[d(G-C)] templates is decreased in the presence of the intercalating agents proflavin, ethidium bromide, acridine orange, ICR-170, and ICR-191. Nearest neighbor analyses of the product of the reaction indicate that two different types of misincorporations occur in the presence of intercalating agents, frameshifts, and single-base substitutions. With alternating polynucleotide templates, frameshifts involving pyrimidines are the most frequent change in sequence observed. Overall, frameshift misincorporations occur with frequencies of one complementary pyrimidine for each intercalated site and one noncomplementary pyrimidine for each 150 sites. From analysis of nearest neighbor frequencies in the product, it is inferred that the intercalating agents interact specifically with pyrimidine (3' leads to 5') purine sequences. An analysis of ratios of correct nucleotide incorporations as a function of intercalator concentration indicates that frameshifts are predominantly additions; however, one cannot rule out infrequent deletions. Base substitutions in the presence of intercalators occur less frequently than frameshifts. From the results of reaction kinetics and nearest neighbor frequencies, it is concluded that the noncomplementary nucleotides are incorporated in phosphodiester linkage and are present as single-base substitutions. Taken together, the results of these studies suggest at least two different modes of action for intercalating agents on the accuracy of DNA synthesis: one leading to frameshift misincorporations and the other leading to single-base substitutions.

Highlights

The effects of intercalating agents on the fidelity of DNA synthesis in vitm have been investigated
Incorporation of Noncomplementary Nucleotides-In order to determine the effects of intercalators on base substitutions duringDNA synthesis,the incorporation of a-32P-labeled complementary nucleotides and 3H-labeled noncomplementary nucleotides was measured simultaneously
The effect of the intercalating agents, proflavin, ethidium bromide, ICR-170, ICR-191a, nd acridine orange on the fidelity of DNA synthesis with the poly[d(A-T)] and poly[d(G-C)]templates using E . coli polymerase I is presented in Tables I and 11

Summary

RESULTS

The effects of intercalators on the accuracy of in uitro DNA synthesis was studied by measuring the frequency of misincorporation of noncomplementary nucleotides and by studying the distribution of complementary and noncomplementary nucleotides by nearest neighbor analysis. The effect of the intercalating agents, proflavin, ethidium bromide, ICR-170, ICR-191a, nd acridine orange on the fidelity of DNA synthesis with the poly[d(A-T)] and poly[d(G-C)]templates using E . In the absence of tion of complementary nucleotides (Table 111).Incorporation incubation, incorporation of dGTP and dATP was I and 0.01 pmol, respectively. Absence of ethidium bromide, the complementary nucleotide The occurrence of TpT sequences is a clear indication of dATP is incorporated in phosphodiester linkage, with greater either transversions, deletions of dAMP, or additions of dTMP than 99%occurring in juxtaposition to dTMP(listed in Table as a result of ethidium bromide. Requirements for the incorporation of complementary and corporation may represent additionsbetween TpA sequences noncomplementary nucleotides or single-base substitutions, dGTP incorporated in place of Reactions werecarried out as given under “Experimental Procedures” with the indicated omissions. There is an increase in 50 ~ L Mproflavin Control -Mg2’ -Poly[d(A-T)] -dTTP -E. coli polymerase I

50 JLM ICR-170

DISCUSSION

C W Shearman and L A Loeb