Depurination decreases fidelity of DNA synthesis in vitro

Abstract

DEPURINATION of DNA results from the breakage of the glycosidic bond between the purine base and the deoxyribose moiety of the purine nucleotides without disrupting the structural integrity of the phosphodiester backbone. Measurements of the rates of depurination of both synthetic polynucleotides and natural DNA in various conditions in vitro suggest that this damage to DNA is a frequent cellular event1. The in vivo rate constant has been estimated1 to be of the order of 1.8 × 10−9min−1. Alkylation2,3 and specific glycosidases4,5 increase the depurination rate constants of those altered bases to 0.08–1.4 × 10−3 and 4.0 × 10−3, respectively. We have therefore been concerned with the possible consequences which may result during replication, repair or transcription when the template contains unrepaired apurinic sites. The biological effects of depurination are not clear. Depurination of DNA has been equated with strand breakage, a potentially lethal lesion6,7, but other evidence indicates that the apurinic site may be quite stable in cells with a half life of up to several months8,9. It is possible, therefore, that depurination of DNA can be a mutagenic event. Mutagenesis by depurination might occur either by an insertion of an incorrect nucleotide opposite the apurinic site or by a deletion at that point during DNA replication or repair. In view of the potentially large number of apurinic sites and their apparent stability, we investigated the effects of depurination of DNA on the fidelity of DNA synthesis in vitro. Recent reports suggest that modification of polynucleotide or DNA templates by various agents, including chemical carcinogens and ultraviolet irradiation, may affect the fidelity of DNA synthesis10–12. We report here that depurination of the synthetic polynucleotide polyd(A-T) results in a decrease in the fidelity of DNA synthesis in vitro using the DNA polymerase from avian myeloblastosis virus (AMV). This is the first demonstration of a possible relationship between depurination and mutagenesis through incorporation of non-complementary nucleotides during DNA synthesis in vitro.