A structural model for sequence-specific proflavin-DNA interactions during in vitro frameshift mutagenesis.

Molecular models describing intermediates that may lead to proflavin-induced 1 bp deletions during in vitro polymerization by E. coli DNA polymerase I Klenow fragment are proposed. The models provide structural explanations for the fact that the induced frameshifts always occur opposite template bases that are adjacent to 5' pyrimidines and are based on the underlying hypothesis that the deletions arise because the polymerase passes by a template base without copying it.

Effects of proflavin and photoactivated proflavin on the template function of single-stranded DNA.

DNA context-specific effects of the association of proflavin, single-stranded DNA and DNA polymerase on DNA polymerization reactions were examined. Frameshift mutations induced by the presence of proflavin during in vitro DNA replication of a single-stranded DNA template by the Klenow fragment of Escherichia coli DNA polymerase I were sequenced. More than 80% of the frameshifts were one base-pair deletions opposite purine bases that were immediately 3' to pyrimidines.

Utilization of 1,N6-etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli.

To test whether vinyl chloride-induced mutagenesis might involve ambiguous base pairing of 1,N6-etheno-adenine (epsilon A) during DNA synthesis, we examined the base pairing potential of epsilon dATP during DNA synthesis catalyzed by Escherichia coli DNA polymerase I (Klenow fragment). An electrophoretic assay of chain elongation was used to assess the degree to which epsilon dATP could substitute for each of the normal dNTPs during elongation of a primer annealed to a bacteriophage template. Despite the fact that the etheno bridge completely blocks normal Watson-Crick pairing of epsilon A with T, we observed that epsilon dATP could substitute for dATP during primer elongation (although inefficiently).

High-performance liquid chromatographic purification of deoxynucleoside 5'-triphosphates and their use in a sensitive electrophoretic assay of misincorporation during DNA synthesis.

This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2'-deoxynucleoside 5'-triphosphates (dNTPs). The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. Anion-exchange HPLC was used to purify the four normal dNTPs (dATP, dGTP, dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N6-etheno-dATP (epsilon dATP).

Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP.