DNA-SEQUENCE EFFECTS ON SINGLE-BASE DELETIONS ARISING DURING DNA POLYMERIZATION IN-VITRO BY ESCHERICHIA-COLI KLENOW FRAGMENT POLYMERASE

Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fra gment are opposite Pu in the template. The most frequent mutations wer e previously found to be associated with the consensus template contex t 5'-PyTPu-3'. In this study, the predictive power of the consensus se quence on single base deletion frequencies was directly tested by para llel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-T TGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deleti ons opposite its neighboring A were never detected. Based on the predi ctions of the consensus, the substitution of T for G should produce fr equent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicte d that deletions opposite template C would be lower than those opposit e either A or G at the same site and this prediction was also fulfille d. The C substitution also produced a new hotspot for 1 bp deletions 1 4 bp away. The new hotspot depends on quasi-palindromic misalignment o f the newly synthesized DNA strand during polymerization; accurate, bu t ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previousl y been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substituti on in the template is consistent with the singular ability of C at tha t site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.