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首页> 外文期刊>Biochemistry >Using a fluorescent cytosine analogue tC° to probe the effect of the Y567 to Ala substitution on the preinsertion steps of dNMP incorporation by RB69 DNA polymerase
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Using a fluorescent cytosine analogue tC° to probe the effect of the Y567 to Ala substitution on the preinsertion steps of dNMP incorporation by RB69 DNA polymerase

机译：使用荧光 胞嘧啶类似物 的tC ° 探测 Y567 的 替代为Ala 上 dNMP的 掺入 的 preinsertion 步骤 由 RB69 DNA聚合酶 的效果

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摘要

Residues in the nascent base pair binding pocket (NBP) of bacteriophage RB69 DNA polymerase (RB69pol) are responsible for base discrimination. Replacing Tyr567 with Ala leads to greater flexibility in the NBP, increasing the probability of misincorporation. We used the fluorescent cytosine analogue, 1,3-diaza-2-oxophenoxazine (tC°), to identify preinsertion step(s) altered by NBP flexibility. When tC° is the templating base in a wild-type (wt) RB69pol ternary complex, its fluorescence is quenched only in the presence of dGTP. However, with the RB69pol Y567A mutant, the fluorescence of tC° is also quenched in the presence of dATP. We determined the crystal structure of the dATP/tC°-containing ternary complex of the RB69pol Y567A mutant at 1.9 ? resolution and found that the incoming dATP formed two hydrogen bonds with an imino-tautomerized form of tC°. Stabilization of the dATP/tC° base pair involved movement of the tC° backbone sugar into the DNA minor groove and required tilting of the tC° tricyclic ring to prevent a steric clash with L561. This structure, together with the pre-steady-state kinetic parameters and dNTP binding affinity, estimated from equilibrium fluorescence titrations, suggested that the flexibility of the NBP, provided by the Y567 to Ala substitution, led to a more favorable forward isomerization step resulting in an increase in dNTP binding affinity. (Chemical Presented).
机译：噬菌体碱基对的残留物中的噬菌体RB69 DNA聚合酶（RB69Pol）的粘合剂袋（NBP）负责基础辨别。用ALA取代TYR567导致NBP的更大灵活性，提高了MISINCORANTION的概率。我们使用荧光胞嘧啶类似物，1,3- diaza-2-氧代苯恶嗪（Tc°），以鉴定通过Nbp柔韧性改变的预介质步骤。当TC°是野生型（WT）RB69Pol三元复合物中的模板碱时，其荧光仅在DGTP存在下淬灭。然而，利用RB69Pol Y567A突变体，TC°的荧光也在DATP存在下淬灭。我们确定了1.9的RB69Pol Y567A突变体的DATP / TC°的晶体结构。分辨率并发现该进入DATP形成了两个氢键，其具有TC°的亚氨基 - 互变异的形式。 DATP / TC°碱基对的稳定化涉及TC°骨架糖的运动进入DNA较小凹槽，需要倾斜TC°三环环，以防止具有L561的空间冲突。这种结构与预稳态的动力学参数和DNTP结合亲和力一起估计来自平衡荧光滴定，表明NBP的灵活性由Y567提供给ALA替代，导致了更有利的前后异构化步骤DNTP结合亲和力的增加。 （呈现化学品）。

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  • 来源
    《Biochemistry》 |2012年第22期|共9页
  • 作者

    Xia S.; Beckman J.; Wang J.; Konigsberg W.H.;

  • 作者单位

    SHM CE-14 Department of Molecular Biophysics and Biochemistry Yale University New Haven CT 06520-8114 United States;

    SHM CE-14 Department of Molecular Biophysics and Biochemistry Yale University New Haven CT 06520-8114 United States;

    SHM CE-14 Department of Molecular Biophysics and Biochemistry Yale University New Haven CT 06520-8114 United States;

    SHM CE-14 Department of Molecular Biophysics and Biochemistry Yale University New Haven CT 06520-8114 United States;

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  • 正文语种 eng
  • 中图分类 生物化学;
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