McCulloch Scott D, Kunkel Thomas A
Laboratory for Molecular Genetics, NIEHS-NIH, North Carolina, USA.
Methods Enzymol. 2006;408:341-55. doi: 10.1016/S0076-6879(06)08021-9.
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.
本文描述了一种测量在合成寡核苷酸引物模板上特定序列中DNA损伤处复制保真度的方法。DNA产物是完整的损伤绕过反应的结果，即含有所有四种脱氧核苷三磷酸，并且既需要在损伤处对面进行插入，又需要从包含损伤的所得引物末端进行多次延伸。新生链被回收并与M13mp2基因组的lacZα互补基因的缺口区域杂交。当将此DNA导入大肠杆菌时，通过M13噬菌斑颜色检测跨损伤DNA合成过程中产生的错误。对突变噬菌斑的DNA进行测序可确定错误类型，并允许计算碱基替换、插入和缺失的错误率。本文举例说明了人DNA聚合酶η对顺式胸腺嘧啶 - 胸腺嘧啶二聚体的绕过。该测定法可用于各种序列背景下的其他损伤，以及用于有或没有辅助蛋白的其他聚合酶。
