Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA.
Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA.
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DOI：
10.1039/c3pp50078k
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发表时间：
2013-08
期刊：
影响因子：
--
通讯作者：
Kohler B
中科院分区：
文献类型：
--
作者：
Law YK;Forties RA;Liu X;Poirier MG;Kohler B
研究了254 nm辐射下两个247碱基对DNA序列中23个胸腺嘧啶-胸腺嘧啶碱基步的胸腺嘧啶-胸腺嘧啶环丁烷嘧啶二聚体（TT-CPD）形成动力学。通过检测由T4嘧啶二聚体糖基化酶在CPD位点切割的片段的5 '-末端处可区分的荧光标记的发射，在正向和反向链上同时位点特异性地测定损伤，并通过凝胶电泳分离。近1000个碱基的总DNA链长度使得有可能监测XTTY类型的所有9个四分体的损伤，其中X和Y是非胸腺嘧啶碱基。TT-CPD产量为不同的四分体被发现变化多达一个数量级，但相似的产量在所有情况下观察到一个给定的四分体。CPD形成在23个不同的网站的动力学分析表明，无论是形成和逆转的光反应敏感地依赖于最近邻的光反应性TT基地的步骤的5'和3'侧的基地的身份。最低的形成和逆转率发生时，两个嘌呤碱基侧翼TT步骤，而最高的形成和逆转率观察到的四分体与至少一个侧翼C。总体而言，结果表明，CPD形成和光反转的概率主要取决于与最近邻基地的相互作用。
The kinetics of thymine-thymine cyclobutane pyrimidine dimer (TT-CPD) formation was studied at 23 thymine-thymine base steps in two 247-base pair DNA sequences irradiated at 254 nm. Damage was assayed site-specifically and simultaneously on both the forward and reverse strands by detecting emission from distinguishable fluorescent labels at the 5’-termini of fragments cleaved at CPD sites by T4 pyrimidine dimer glycosylase and separated by gel electrophoresis. The total DNA strand length of nearly 1000 bases made it possible to monitor damage at all 9 tetrads of the type XTTY, where X and Y are non-thymine bases. TT-CPD yields for different tetrads were found to vary by as much as an order of magnitude, but similar yields were observed at all instances of a given tetrad. Kinetic analysis of CPD formation at 23 distinct sites reveals that both the formation and reversal photoreactions depend sensitively on the identity of the nearest-neighbour bases on the 5’ and 3’ side of a photoreactive TT base step. The lowest formation and reversal rates occur when two purine bases flank a TT step, while the highest formation and reversal rates are observed for tetrads with at least one flanking C. Overall, the results show that the probabilities of CPD formation and photoreversal depend principally on interactions with nearest-neighbour bases.
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