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首页> 外文期刊>Biochemistry >Ambivalent Incorporation of the Fluorescent Cytosine Analogues tC and tCo by Human DNA Polymerase α and Klenow Fragment
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Ambivalent Incorporation of the Fluorescent Cytosine Analogues tC and tCo by Human DNA Polymerase α and Klenow Fragment

机译：荧光胞嘧啶类似物tC和tCo被人DNA聚合酶α和Klenow片段双价掺入

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摘要

Westudied the incorporation of the fluorescent cytidine analogues 1,3-diaza-2-oxophenothiazine (tC)nand 1,3-diaza-2-oxophenoxazine (tCo) by human DNA polymerase R and Klenow fragment of DNAnpolymerase I (Escherichia coli). These tricyclic nucleobases possess the regular hydrogen bonding interface ofncytosine but are significantly expanded in size toward the major groove. Despite the size alteration, both DNAnpolymerases insert dtCTP and dtCoTP with remarkable catalytic efficiency. Polymerization opposite guanine isncomparable to the insertion of dCTP, while the insertion opposite adenine is only ∼4-11 times less efficient thannthe formation of a T-A base pair. Both enzymes readily extend the formed tC(o)-G and tC(o)-A base pairs andncan incorporate at least four consecutive nucleotide analogues. Consistent with these results, both DNAnpolymerases efficiently polymerize dGTP and dATP when tC and tCo are in the template strand. Klenownfragment inserts dGTP with a 4-9-fold higher probability than dATP, while polymerase R favors dGTP overndATP by a factor of 30-65. Overall, the properties of tC(o) as a templating base and as an incoming nucleotidenare surprisingly symmetrical and may be universal for A and B family DNA polymerases. This finding suggestsnthat the aptitude for ambivalent base pairing is a consequence of the electronic properties of tC(o).
机译：研究了人DNA聚合酶R和DNAn聚合酶I（大肠杆菌）的Klenow片段掺入荧光胞苷类似物1，3-二氮杂-2-氧代吩噻嗪（tC）n和1，3-二氮杂-2-氧代吩恶嗪（tCo）。这些三环核碱基具有胞嘧啶的规则氢键界面，但在大小上朝着主沟明显扩展。尽管大小发生变化，但两种DNA聚合酶均以显着的催化效率插入dtCTP和dtCoTP。与鸟嘌呤相反的聚合与dCTP的插入无可比拟，而与腺嘌呤相反的插入的效率仅比形成T-A碱基对低约4-11倍。两种酶都容易扩展形成的tC（o）-G和tC（o）-A碱基对，并且可以掺入至少四个连续的核苷酸类似物。与这些结果一致，当tC和tCo在模板链中时，两种DNA聚合酶均有效地聚合了dGTP和dATP。 Klenownfragment插入dGTP的可能性比dATP高4-9倍，而聚合酶R则使dGTP胜过ndATP 30-65倍。总体而言，tC（o）作为模板碱基和传入核苷酸的性质令人惊讶地对称，并且可能是A和B家族DNA聚合酶通用的。这一发现表明，矛盾碱基配对的能力是tC（o）电子性质的结果。

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    《Biochemistry》 |2009年第31期|p.7547-7555|共9页
  • 作者

    Gudrun Stengel Byron W. Purse L. Marcus Wilhelmsson Milan Urban and Robert D. Kuchta;

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    ‡Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 30309-0215, §Department of Chemistry andBiochemistry, University of Denver, Denver, Colorado 80208, and)Department of Chemical and Biological Engineering/PhysicalChemistry, Chalmers University of Technology, SE-41296 Gothenburg, Sweden;

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