Klenow fragment-DNA interaction required for the incorporation of nucleotides opposite guanine and O6-methylguanine.
Klenow fragment-DNA interaction required for the incorporation of nucleotides opposite guanine and O6-methylguanine.
复制标题
与鸟嘌呤和 O6-甲基鸟嘌呤相反的核苷酸掺入所需的 Klenow 片段-DNA 相互作用。
DOI：
10.1021/bi971253g
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Spratt,TE
中科院分区：
文献类型：
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作者：
Spratt,TE
DNA聚合酶I的Klenow片段监测碱基对几何形状的机制可能涉及聚合酶和新生碱基对的小凹槽之间的氢键。通过合成含有鸟嘌呤和3-去氮鸟嘌呤的寡脱氧核苷酸，并比较四种dNTPs结合的稳态动力学，研究了鸟嘌呤在模板链中的3位的参与。只有当dCTP为共底物时，vmax / km显著降低（170倍），这表明只有在正确的碱基对被复制时才存在氢键。该方法还用于研究Klenow片段如何与致突变碱基O6mG的3位相互作用。dTTP对物o6 -甲基-3-脱氮鸟嘌呤（O6m3DG）掺入的vmax / km比对物o6mg低1700倍。相比之下，相对于toO6mG，与eo6m3dg相反的dCTP掺入后，inVmax/ kmoc增加了6倍。这一结果表明，Klenow片段与do6mg之间的氢键在与o6mg相反的dTTP结合中更为重要，并可能与o6mg的致突变性有关。
A mechanism by which the Klenow fragment of DNA polymerase I monitors the geometry of the base pairs may involve hydrogen bonds between the polymerase and the minor groove of the nascent base pair. The involvement of the 3-position of guanine in the template strand was examined by synthesizing oligodeoxynucleotides containing guanine and 3-deazaguanine and comparing the steady-state kinetics of the incorporation of all four dNTPs. TheVmax/Kmdecreased a significant amount (170-fold) only when dCTP was the co-substrate suggesting that a hydrogen bond exists only when the correct base pair is being replicated. This approach was also used to examine how the Klenow fragment interacts with the 3-position of the mutagenic baseO6-methylguanine (O6mG). TheVmax/Kmfor the incorporation of dTTP oppositeO6-methyl-3-deazaguanine (O6m3DG) was 1700-fold less than oppositeO6mG. In contrast, a small 6-fold increase inVmax/Kmoccurred for the incorporation of dCTP oppositeO6m3DG relative toO6mG. This result suggests that the hydrogen bond between the Klenow fragment andO6mG is more important in the incorporation of dTTP oppositeO6mG and may contribute to the mutagenicity ofO6mG.