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首页> 外文期刊>Biochemistry >TARGETED BASE STACKING DISRUPTION BY THE ECORI DNA METHYLTRANSFERASE
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TARGETED BASE STACKING DISRUPTION BY THE ECORI DNA METHYLTRANSFERASE

机译：ECORI DNA甲基转移酶的靶向碱基堆积破坏

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摘要

We describe a novel fluorescence-based assay for detecting DNA conformational alterations within enzyme-DNA complexes. The target adenine for EcoRI DNA methyltransferase (GA(A)TTC) was replaced with 2-aminopurine, which fluoresces upon excitation at 310 nm. Addition of the methyltransferase to the duplex binding site results in a 14-fold increase in fluorescence intensity with a 10 nm blue shift. The fluorescence is similar to 50% of that observed with equimolar free nucleoside, consistent with extrahelical stabilization of the target base in the enzyme-DNA complex. The shift in lambda(max) further implies the base is placed into a low dielectric environment. For adenine-specific DNA methyltransferases, a hydrophobic pocket composed of highly conserved amino acids lies proximal to the cofactor binding site. Substitution of 2-aminopurine adjacent to the target base also results in detectable changes in fluorescence emission following complex formation with the methyltransferase. Thus, other classes of enzymes hypothesized to utilize base flipping can be investigated by this method.
机译：我们描述了一种新型的基于荧光的检测酶-DNA复合物中DNA构象变化的检测方法。 EcoRI DNA甲基转移酶（GA（A）TTC）的目标腺嘌呤被2-氨基嘌呤取代，后者在310 nm激发时发出荧光。将甲基转移酶添加到双链体结合位点导致荧光强度增加14倍，蓝移为10 nm。荧光类似于在等摩尔游离核苷中观察到的荧光，与酶-DNA复合物中靶碱基的螺旋外稳定相一致。 lambda（max）的变化还意味着该基极被置于低介电环境中。对于腺嘌呤特异性DNA甲基转移酶，由高度保守的氨基酸组成的疏水口袋位于辅因子结合位点的近端。与甲基转移酶形成复合物后，与靶碱基相邻的2-氨基嘌呤的取代也导致荧光发射的可检测变化。因此，可以通过这种方法研究假设利用碱基翻转的其他类型的酶。

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