Nucleotide insertion kinetics opposite abasic lesions in DNA.
Nucleotide insertion kinetics opposite abasic lesions in DNA.
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DOI：
10.1016/s0021-9258(18)48324-x
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发表时间：
1987-05
期刊：
影响因子：
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通讯作者：
S. Randall;Ramon Eritjaj;Bruce E. Kaplanj;John PetruskaS;M. Goodman
中科院分区：
文献类型：
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作者：
S. Randall;Ramon Eritjaj;Bruce E. Kaplanj;John PetruskaS;M. Goodman
引入凝胶测定来测量沿 DNA 模板链单个位点的 DNA 聚合酶插入动力学。该测定用于分析使用果蝇 DNA 聚合酶 α 在合成脱碱基（无嘌呤/无嘧啶）病变对面插入脱氧核苷酸的动力学。无碱基损伤位于不同最近邻碱基旁边，可以评估碱基堆积对插入特异性的影响。核苷酸插入的特异性Vmax/Km，A比G高6-11倍，A比C和T高约20-50倍。无碱基损伤处的插入特异性似乎更多地取决于Vmax的差异而不是Km的差异。插入 A 和 G 脱氧核苷酸的表观米氏常数相似，大约相差 2 倍。当 T 是引物链上的 5'-最近邻居时，A 或 G 的插入在无碱基损伤处发生的效率最高，而当 G 是 5'-最近邻居时，插入效率最低。插入位点附近不同碱基堆积伙伴的存在对特异性的影响高达 4 倍。
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.