Nucleotide insertion kinetics opposite abasic lesions in DNA.
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.
D000818:Animals; D001080:Apurinic Acid; D001483:Base Sequence; D004249:DNA Damage; D004257:DNA Polymerase II / Q000378:metabolism*; D004330:Drosophila / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008957:Models, Genetic; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011119:Polynucleotides; D013698:Templates, Genetic
D009838:Oligodeoxyribonucleotides; D011119:Polynucleotides; C001790:apyrimidinic acid; D001080:Apurinic Acid; D004257:DNA Polymerase II