Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 2200 Pierce Avenue, Nashville, TN 37232-0146, USA.
J Mol Biol. 2010 Feb 5;395(5):1007-18. doi: 10.1016/j.jmb.2009.11.071. Epub 2009 Dec 4.
Translesion DNA polymerases are more efficient at bypass of many DNA adducts than replicative polymerases. Previous work with the translesion polymerase Sulfolobus solfataricus Dpo4 showed a decrease in catalytic efficiency during bypass of bulky N(2)-alkyl guanine (G) adducts with N(2)-isobutylG showing the largest effect, decreasing approximately 120-fold relative to unmodified deoxyguanosine (Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N(2)-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576). The effect of adduct size on individual catalytic steps has not been easy to decipher because of the difficulty of distinguishing early noncovalent steps from phosphodiester bond formation. We developed a mutant with a single Trp (T239W) to monitor fluorescence changes associated with a conformational change that occurs after binding a correct 2'-deoxyribonucleoside triphosphate (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) and, in the present work, utilized this approach to monitor insertion opposite N(2)-alkylG-modified oligonucleotides. We estimated maximal rates for the forward conformational step, which coupled with measured rates of product formation yielded rate constants for the conformational step (both directions) during insertion opposite several N(2)-alkylG adducts. With the smaller N(2)-alkylG adducts, the conformational rate constants were not changed dramatically (<3-fold), indicating that the more sensitive steps are phosphodiester bond formation and partitioning into inactive complexes. With the larger adducts (>or=(2-naphthyl)methyl), the absence of fluorescence changes suggests impaired ability to undergo an appropriate conformational change, consistent with previous structural work.
跨损伤 DNA 聚合酶在绕过许多 DNA 加合物方面比复制聚合酶更有效。先前使用跨损伤聚合酶 Sulfolobus solfataricus Dpo4 的研究表明，在绕过体积较大的 N(2)-烷基鸟嘌呤 (G) 加合物时，催化效率降低，其中 N(2)-异丁基 G 的影响最大，相对于未修饰的脱氧鸟嘌呤，降低了约 120 倍（Zhang, H., Eoff, R. L., Egli, M., Guengerich, F. P. Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translation synthesis past bulky N(2)-alkylguanine adducts. J. Biol. Chem. 2009; 284: 3563-3576）。由于难以区分早期非共价步骤和磷酸二酯键形成，因此很难确定加合物大小对各个催化步骤的影响。我们开发了一种突变体，其中单个色氨酸（T239W）用于监测与结合正确的 2'-脱氧核糖核苷三磷酸 (Beckman, J. W., Wang, Q., Guengerich, F. P. Kinetic analysis of nucleotide insertion by a Y-family DNA polymerase reveals conformational change both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 2008; 283: 36711-36723) 后发生的构象变化相关的荧光变化，并且在本工作中，利用这种方法监测 N(2)-烷基 G 修饰的寡核苷酸的插入。我们估计了正向构象步骤的最大速率，该速率与产物形成的测量速率相结合，得出了在几种 N(2)-烷基 G 加合物的对面插入时构象步骤（两个方向）的速率常数。对于较小的 N(2)-烷基 G 加合物，构象速率常数没有发生显著变化（<3 倍），表明更敏感的步骤是磷酸二酯键形成和分配到非活性复合物中。对于较大的加合物（>=(2-萘基)甲基），荧光变化的缺失表明无法进行适当的构象变化，这与先前的结构研究一致。
