Abstract
A gel assay is introduced to measure DNA polymerase insertion kinetics at single sites along a DNA template strand. The assay is used to analyze the kinetics of inserting deoxynucleotides opposite a synthetic abasic (apurinic/apyrimidinic) lesions using Drosophila DNA polymerase alpha. The location of the abasic lesion next to different nearest-neighbor bases allows the effects of base stacking on the specificity of insertion to be evaluated. The specificity of nucleotide insertion, Vmax/Km, is 6-11 times greater for A over G and about 20-50 times greater for A over C and T. The insertion specificity at the abasic lesion appears to depend more on differences in Vmax than Km. Apparent Michaelis constants for inserting A and G deoxynucleotides are similar to within about a factor of 2. The insertion of A or G occurs most efficiently at the abasic lesion when T is the 5'-nearest neighbor on the primer strand and least efficiently when G is the 5'-nearest neighbor. The presence of different base stacking partners adjacent to the site of insertion has up to a 4-fold effect on specificity.
Highlights
A gel assay is introduced to measureDNA polymer- are theexpected major type of mutation [9]
Depending on the relative importance of a and b, significant differences in V, and/or K, may be seen in the kinetics of Abasic sites are noncoding lesions in DNA that arise by hydrolysis of the glycosidic bond connecting purine or pyrimidine base to deoxyribose sugar
The relative velocity of nucleotide insertion at each substrate concentration is measured as the intensity ratio Zz/Z1,observed on a polyacrylamide gel for 5’-32P-labeledprimers extended to theabasic site (Z2) and one basic prior to the abasic site (Z1).Values of V,”and K,are calculated by a linear least-squares tit to Hanes-Woolf plots for each template
Summary
Depending on the relative importance of a and b, significant differences in V,,, and/or K,,, may be seen in the kinetics of Abasic (apurinic or apyrimidinic) sites are noncoding lesions in DNA that arise by hydrolysis of the glycosidic bond connecting purine or pyrimidine base to deoxyribose sugar. Abasic site would not be contaminated with any of the preceding labeled primer were run on a gel and exposed over 3-h to 2-day time bases. Band intensities were determined by cleotides long; the unlabeled template strand 26 nucleotides long, of integration of areas under band tracingusing the GS350data system which 15 are complementary to primer, and the remainder of the purchased from Hoefer Scientific Instruments.
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