Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides.
Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides.
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DOI：
10.1093/nar/gkn404
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发表时间：
2008-08
影响因子：
14.9
通讯作者：
Imanishi T
中科院分区：
文献类型：
--
作者：
Kuwahara M;Obika S;Nagashima J;Ohta Y;Suto Y;Ozaki H;Sawai H;Imanishi T
为了系统分析糖基核苷修饰在DNA聚合反应中的作用，我们合成了16个含有2‘，4’桥联核苷酸的修饰模板和3种不同桥联结构的2‘，4’桥联核苷-5‘-三磷酸盐。在所用的五种耐热DNA聚合酶Taq、Phusion HF、Vent(exo-)、KOD Dash和KOD(exo-)中，KOD Dash和KOD(exo-)DNA聚合酶能够以几个核苷酸的间隔顺利地阅读含有2‘-O，4’-C-亚甲基连接的核苷酸的修饰模板，即使在标准的酶浓度下也是如此。虽然Vent(exo-)DNA聚合酶也阅读这些修饰的模板，但动力学研究表明，KOD(exo-)DNA聚合酶在准确掺入核苷酸方面远远优于Vent(exo-)DNA聚合酶。当使用任何一种DNA聚合酶时，模板链上2‘，4’桥联核苷酸的存在大大降低了核苷酸掺入的反应速度。含有7个连续的2‘，4’桥联核苷酸序列的修饰模板不能被所使用的任何DNA聚合酶完全转录；更长的加长产物的产率按修饰糖的空间膨胀的顺序递减。使用2‘，4’-桥联核苷-5‘-三磷酸盐将2’，4‘-桥联核苷酸连续掺入延伸链中要困难得多。这些数据表明，当糖与延伸末端相邻时，与碱基修饰一样，糖修饰对聚合酶反应的影响比它也在模板上时更大。
In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2′,4′-bridged nucleotides and three types of 2′,4′-bridged nucleoside-5′-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates containing 2′-O,4′-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min. Although the Vent(exo-) DNA polymerase also read through these modified templates, kinetic study indicates that the KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides. When either of the DNA polymerase was used, the presence of 2′,4′-bridged nucleotides on a template strand substantially decreased the reaction rates of nucleotide incorporations. The modified templates containing sequences of seven successive 2′,4′-bridged nucleotides could not be completely transcribed by any of the DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars. Successive incorporation of 2′,4′-bridged nucleotides into extending strands using 2′,4′-bridged nucleoside-5′-triphospates was much more difficult. These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification.
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影响因子：
15
作者：
Inoue, Naonori;Shionoya, Aki;Matsuda, Akira
通讯作者：
Matsuda, Akira
影响因子：
2.9
作者：
BECKMAN, RA;MILDVAN, AS;LOEB, LA
通讯作者：
LOEB, LA
影响因子：
14.9
作者：
Karthikeyan, G;Chary, KVR;Rao, BJ
通讯作者：
Rao, BJ
影响因子：
5.6
作者：
Hashimoto, H;Nishioka, M;Kai, Y
通讯作者：
Kai, Y
影响因子：
14.9
作者：
CLARK, JM
通讯作者：
CLARK, JM