In vitro mutagenesis in the lacI gene of Escherichia coli: Fate of 3′-terminal mispairs versus internal base mispairs in a transfection assay

Rogelio Maldonado-Rodriguez, Kenneth L. Beattie

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Abstract

The fate of G · T mismatches and frameshifts, present at the 3′-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli. Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G · T mismatches in the middle of the duplex region or at the 3′-OH end of the primer. Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G · T mismatch and an extra nucleotide. The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs ('complete' reaction) or in the absence of dATP ('-A' reaction) was monitored by gel electrophoresis. Duplex DNA products were used in a transfection assay and the nucleotide changes in i- mutant progeny were determined by sequence analysis. The results suggest that whereas a single 3′-terminal G · T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G · T mismatches are degraded by the 3′-exonuclease activity of this polymerase prior to primer extension. This editing activity is increased with the number of 3′-terminal mispairs. Single, double and triple T → C base substitutions were efficiently recovered when the mismatches occurred internally. Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively. When products of the '-A' misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T → C), but no frameshifts, were recovered. The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic 'minus' conditions was due to the efficient action of the 3′-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay. © 1991.
Original languageAmerican English
Pages (from-to)5-18
Number of pages3
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
DOIs
StatePublished - 1 Jan 1991
Externally publishedYes

Fingerprint

mutagenesis
primers
Mutagenesis
spleen exonuclease
Nucleotides
Escherichia
genes
Escherichia coli
Transfection
Assays
DNA
Enzymes
Genes
Substitution reactions
nucleotides
Polymerization
Elongation
Bearings (structural)
enzymes
Bacteriophage M13

Cite this

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title = "In vitro mutagenesis in the lacI gene of Escherichia coli: Fate of 3′-terminal mispairs versus internal base mispairs in a transfection assay",
abstract = "The fate of G · T mismatches and frameshifts, present at the 3′-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli. Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G · T mismatches in the middle of the duplex region or at the 3′-OH end of the primer. Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G · T mismatch and an extra nucleotide. The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs ('complete' reaction) or in the absence of dATP ('-A' reaction) was monitored by gel electrophoresis. Duplex DNA products were used in a transfection assay and the nucleotide changes in i- mutant progeny were determined by sequence analysis. The results suggest that whereas a single 3′-terminal G · T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G · T mismatches are degraded by the 3′-exonuclease activity of this polymerase prior to primer extension. This editing activity is increased with the number of 3′-terminal mispairs. Single, double and triple T → C base substitutions were efficiently recovered when the mismatches occurred internally. Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively. When products of the '-A' misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T → C), but no frameshifts, were recovered. The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic 'minus' conditions was due to the efficient action of the 3′-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay. {\circledC} 1991.",
author = "Rogelio Maldonado-Rodriguez and Beattie, {Kenneth L.}",
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T1 - In vitro mutagenesis in the lacI gene of Escherichia coli: Fate of 3′-terminal mispairs versus internal base mispairs in a transfection assay

AU - Maldonado-Rodriguez, Rogelio

AU - Beattie, Kenneth L.

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N2 - The fate of G · T mismatches and frameshifts, present at the 3′-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli. Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G · T mismatches in the middle of the duplex region or at the 3′-OH end of the primer. Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G · T mismatch and an extra nucleotide. The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs ('complete' reaction) or in the absence of dATP ('-A' reaction) was monitored by gel electrophoresis. Duplex DNA products were used in a transfection assay and the nucleotide changes in i- mutant progeny were determined by sequence analysis. The results suggest that whereas a single 3′-terminal G · T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G · T mismatches are degraded by the 3′-exonuclease activity of this polymerase prior to primer extension. This editing activity is increased with the number of 3′-terminal mispairs. Single, double and triple T → C base substitutions were efficiently recovered when the mismatches occurred internally. Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively. When products of the '-A' misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T → C), but no frameshifts, were recovered. The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic 'minus' conditions was due to the efficient action of the 3′-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay. © 1991.

AB - The fate of G · T mismatches and frameshifts, present at the 3′-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli. Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G · T mismatches in the middle of the duplex region or at the 3′-OH end of the primer. Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G · T mismatch and an extra nucleotide. The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs ('complete' reaction) or in the absence of dATP ('-A' reaction) was monitored by gel electrophoresis. Duplex DNA products were used in a transfection assay and the nucleotide changes in i- mutant progeny were determined by sequence analysis. The results suggest that whereas a single 3′-terminal G · T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G · T mismatches are degraded by the 3′-exonuclease activity of this polymerase prior to primer extension. This editing activity is increased with the number of 3′-terminal mispairs. Single, double and triple T → C base substitutions were efficiently recovered when the mismatches occurred internally. Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively. When products of the '-A' misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T → C), but no frameshifts, were recovered. The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic 'minus' conditions was due to the efficient action of the 3′-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay. © 1991.

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