Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
Nucleic Acids Res. 2011 Mar;39(4):1623-37. doi: 10.1093/nar/gkq925. Epub 2010 Oct 15.
This report examines the molecular mechanism by which high-fidelity DNA polymerases select nucleotides during the replication of an abasic site, a non-instructional DNA lesion. This was accomplished by synthesizing several unique 5-substituted indolyl 2'-deoxyribose triphosphates and defining their kinetic parameters for incorporation opposite an abasic site to interrogate the contributions of π-electron density and solvation energies. In general, the K(d, app) values for hydrophobic non-natural nucleotides are ∼10-fold lower than those measured for isosteric hydrophilic analogs. In addition, k(pol) values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density. The differences in kinetic parameters were used to quantify the energetic contributions of desolvation and π-electron density on nucleotide binding and polymerization rate constant. We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site. Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.
本报告探讨了高保真 DNA 聚合酶在复制碱基缺失位点（一种无指令性的 DNA 损伤）时选择核苷酸的分子机制。通过合成几种独特的 5-取代吲哚基 2'-脱氧核糖三磷酸，并定义它们在碱基缺失位点处掺入的动力学参数，以研究 π 电子密度和溶剂化能的贡献。一般来说，疏水性非天然核苷酸的 K(d,app) 值比等电子亲水性类似物低约 10 倍。此外，π 电子密度较低的核苷酸的 k(pol) 值比具有更高 π 电子密度的等电子类似物慢。动力学参数的差异用于量化核苷酸结合和聚合速率常数中去溶剂化和 π 电子密度的能量贡献。我们证明，缺乏氢键能力的类似物作为跨损伤 DNA 复制的链终止子，而具有氢键官能团的类似物在配对到碱基缺失位点时会被延伸。总的来说，数据表明，碱基缺失位点处核苷酸的掺入效率受与核碱基去溶剂化和 π 电子堆积相互作用相关的能量控制，而延伸到损伤之外则是通过碱基堆积和氢键相互作用的结合来实现的。
