Base analog N6-hydroxylaminopurine mutagenesis in Escherichia coli: genetic control and molecular specificity.

Abstract:

We have studied the molecular specificity of the base analog N6-hydroxylaminopurine (HAP) in the E. coli lacI gene, as well as the effects of mutations in DNA repair and replication genes on HAP mutagenesis. HAP induced base substitutions of the two transition types (A . T-->G . C and G . C-->A . T) at equal frequency. This bi-directional transition specificity is consistent with in vitro primer extension experiments with the Klenow fragment of DNA polymerase I in which we observed that either dTTP or dCTP were incorporated opposite HAP in an oligonucleotide template. The spectrum of HAP-induced transitions was different from the spontaneous transitions in either a wild-type or a mismatch-repair-defective (mutL) strain. Mutations in genes controlling excision repair, exonucleolytic proofreading, mismatch correction, error-prone (SOS) repair and 8-oxo-guanine repair did not affect HAP-induced mutagenesis substantially. However, an extensive deletion of several genes in the uvrB-bio region conferred supersensitivity to the lethal and mutagenic effects of HAP, perhaps due to an effect on HAP metabolism. dnaE antimutator alleles reduced HAP-forward mutagenicity in allele-specific manner: dnaE911 reduced it several fold, while dnaE915 abolished it almost completely. The results obtained are consistent with the idea that HAP is mutagenic in E. coli via a pathway generating replication errors.

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