Effects of depurination on the fidelity of DNA synthesis

Abstract

The effect of depurination of polynucleotide templates on the fidelity of DNA synthesis in vitro has been determined. The fidelity of DNA synthesis with Escherichia coli DNA polymerase I, avian myeloblastosis virus DNA polymerase and human placenta DNA polymerase-β is decreased as a result of depurination of the poly[d(A-T)], poly[d(G-C)]and poly[d(A)]templates. The error rate with poly[d(A-T)]increased from 1 17,500 to 1 2100 using E. coli Pol I, and from 1 4100 to 1 1500 using the myeloblastosis virus DNA polymerase. Depurination of poly[d(A)]increased the error rate from 1 21,000 to 1 6500 using E. coli Pol I, and from 1 19,300 to 1 6100 using the DNA polymerase-β from human placenta. Depurination of poly[d(G-C)]resulted in an increase in the error rate with E. coli Pol I from 1 9200 to 1 2200 , and with the virus DNA polymerase from 1 2400 to 1 1300 . This misincorporation is shown to be directly proportional to the extent of depurination. Deletion experiments and alkaline sucrose gradient analyses suggest that the incorporation of complementary and non-complementary nucleotides is dependent on polymerization, and occurs in the same newly synthesized product. Kinetic studies and nearest-neighbor analyses indicate that the incorporation of non-complementary nucleotides occurs randomly as single-base substitutions. The nearest-neighbor studies also suggest that any of the four deoxynucleotides can be incorporated opposite apurinic sites. The number of each nucleotide incorporated relative to the number of apurinic sites was determined to be 1 490 for dGTP, 1 115 for dCTP, 1 2·5 for dATP and 1 1·7 for dTTP with both the poly[d(A-T)] and poly[d(A)] templates. The frequencies of misincorporation relative to the number of apurinic sites with the poly[d(G-C)]template were 1 230 for dATP, 1 120 for dTTP, 1 2·4 for dGTP and 1 1·8 for dCTP. Hydrolysis at the apurinic sites by alkali treatment reversed the effects of depurination on fidelity. The error rates with the depurinated templates were reduced to within 2% of those obtained prior to depurination, providing additional evidence that the misincorporation after depurination results from apurinic sites on the template. These results suggest a possible relationship between depurination of DNA and errors in DNA replication and/or repair.