Measuring the fidelity of translesion DNA synthesis.
A method is described to measure the fidelity of copying past a DNA lesion in a defined sequence on a synthetic oligonucleotide primer-template. The DNA product is the result of a complete lesion bypass reaction, i.e., containing all four deoxynucleotide triphosphates and requiring both insertion opposite the lesion and multiple extensions from the resulting primer termini containing the lesion. The nascent strand is recovered and hybridized to a gapped region of the lacZalpha complementation gene of the M13mp2 genome. When this DNA is introduced into Escherichia coli, errors made during translesion DNA synthesis are detected by M13 plaque colors. Sequencing of DNA from mutant plaques defines the types of errors and permits calculation of error rates for base substitutions, insertions, and deletions. The method is illustrated here for bypass of a cis-syn thymine-thymine dimer by human DNA polymerase eta. The assay can be used with other lesions in various sequence contexts and with other polymerases with or without accessory proteins.
McCulloch
Scott D
SD
Laboratory for Molecular Genetics, NIEHS-NIH, North Carolina, USA.
Kunkel
Thomas A
TA
D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D011740:Pyrimidine Dimers; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D009841:Oligonucleotides; D011740:Pyrimidine Dimers; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases