title: DNA sequence effects on single base deletions arising during DNA polymerization in vitro by Escherichia coli Klenow fragment polymerase.
abstract: Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent mutations were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent 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 predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously 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 substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.
authors.affiliation: Department of Microbiology and Molecular Biology, UMD-New Jersey Medical School, Newark 07103.
authors.forename: F J
authors.initials: FJ
authors.lastname: Wang
authors.forename: L S
authors.initials: LS
authors.lastname: Ripley
mesh_terms: D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000096:biosynthesis* / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism*; D015342:DNA Probes / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D016368:Frameshift Mutation; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017384:Sequence Deletion
chemical_list: D015342:DNA Probes; D004247:DNA; D004256:DNA Polymerase I