Luo Guobin, Wang Mina, Konigsberg William H, Xie X Sunney
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12610-5. doi: 10.1073/pnas.0700920104. Epub 2007 Jul 18.
We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol ternary complex upon binding of a dNTP substrate. This fluorescence change is believed to reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function. The rate of the conformational change induced by a complementary dNTP substrate was determined by both conventional stopped-flow and high-time-resolution continuous-flow fluorescence measurements at the ensemble-averaged level. The rate of this conformational change is much faster than that of DNA synthesis but is significantly reduced for noncomplementary dNTPs, as revealed by single-molecule measurements. The high level of selectivity of incoming dNTPs pertinent to this conformational change is a major contributor to replicative fidelity.
我们报告了针对噬菌体T7 DNA聚合酶（T7 pol）功能上重要的构象变化的荧光测定法，该方法利用附着于DNA底物上的Cy3染料的环境敏感性。在单分子水平上观察到Cy3荧光强度增加，这反映了在结合dNTP底物时T7 pol三元复合体内的构象变化。这种荧光变化被认为反映了T7 pol指状结构域的闭合，这对聚合酶功能至关重要。在整体平均水平上，通过传统的停流和高时间分辨率连续流荧光测量来确定由互补dNTP底物诱导的构象变化速率。单分子测量表明，这种构象变化的速率比DNA合成的速率快得多，但对于非互补dNTP则显著降低。与这种构象变化相关的进入dNTP的高选择性水平是复制保真度的主要贡献因素。
