Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.
Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.
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DOI：
10.1093/nar/gkp266
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发表时间：
2009-07
影响因子：
14.9
通讯作者：
Wilhelmsson LM
中科院分区：
文献类型：
--
作者：
Sandin P;Stengel G;Ljungdahl T;Börjesson K;Macao B;Wilhelmsson LM
DNA聚合酶选择正确核苷酸的机制研究经常使用荧光标记的DNA来监测聚合酶-DNA复合体对进入的核苷酸的反应的构象重排。为此，荧光碱基类似物发挥着越来越重要的作用，因为它们对DNA-蛋白质相互作用的干扰比连接的荧光团少。在这里，我们报道了两个特别明亮的胞嘧啶类似物1，3-二氮-2-氧代吩噻嗪(TC)及其氧代同系物1，3-二氮-2-氧代吩嗪(TCO)的5‘-三磷酸通过Klenow片段掺入DNA。根据荧光各向异性测量，这两个核苷酸类似物相对于鸟嘌呤的聚合效率比胞嘧啶三磷酸略高，并被证明与DNA聚合酶活性部位具有纳摩尔亲和力。使用这种方法，我们进行了竞争性结合实验，并表明它们可以用来确定任何给定的天然或非天然核苷酸的解离常数。结果表明，Klenow片段的活性中心足够灵活，可以容忍在主槽中尺寸扩大的碱基对。此外，酶法高效聚合荧光核苷酸的可能性补充了可用于研究DNA复制的生物物理探针工具箱。
Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase–DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA–protein interaction than do tethered fluorophores. Here we report the incorporation of the 5′-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tCO), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication.
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影响因子：
3.2
作者：
Chelliserrykattil, Jijumon;Lu, Haige;Lee, Alex H. F.;Kool, Eric T.
通讯作者：
Kool, Eric T.
影响因子：
4.7
作者：
Ikeda, Shuji;Kubota, Takeshi;Okamoto, Akimitsu
通讯作者：
Okamoto, Akimitsu
影响因子：
2.9
作者：
Lee, Benjamin J.;Barch, Maryan;Breslauer, Kenneth J.
通讯作者：
Breslauer, Kenneth J.
影响因子：
2.9
作者：
Hawkins, ME;Pfleiderer, W;Knutson, JR
通讯作者：
Knutson, JR
影响因子：
2.9
作者：
BLOOM, LB;OTTO, MR;GOODMAN, MF
通讯作者：
GOODMAN, MF