Structure and dynamics in DNA looped domains:: CAG triplet repeat sequence dynamics probed by 2-aminopurine fluorescence
Structure and dynamics in DNA looped domains:: CAG triplet repeat sequence dynamics probed by 2-aminopurine fluorescence
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DOI：
10.1021/bi7005674
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发表时间：
2007-09-25
期刊：
影响因子：
2.9
通讯作者：
Breslauer, Kenneth J.
中科院分区：
文献类型：
--
作者：
Lee, Benjamin J.;Barch, Maryan;Breslauer, Kenneth J.
三联体重复序列（CAG）（n）和相关三联体重复序列与涉及许多使人衰弱的疾病的动态DNA突变相关。为了深入了解（CAG）（n）重复序列的动力学，我们在（GC）（3）（CAG）（6）（GC）（3）发夹寡核苷酸的18个碱基环状结构域内的选定位置用单个2-氨基嘌呤（2AP）荧光碱基取代腺嘌呤。使用温度依赖性稳态荧光测量结合时间相关光子计数光谱，我们显示的构象和动力学的C2 APG域是强烈依赖于探针的位置在环状区域。换句话说，（CAG）6三联体重复环状结构域不是均匀的单链环，而是表现出位置依赖性的顺序和动力学。C2 APG重复序列内的2AP荧光动力学由4组分指数衰减模型很好地描述，寿命范围为5 ps至4 ns。全局DNA构象（双链体、发夹、单链）以及探针在给定发夹环内的局部位置的差异主要反映在相对振幅而不是探针的寿命中。发夹（CAG）（n）环中的时间依赖性2AP各向异性对荧光碱基的位置敏感，其中位于环内中心的2AP探针的荧光去极化比位于环-茎连接附近的重复序列的5 '或3'末端的2AP进行得显著更慢。这些结果与CAG重复序列的节段性运动一致，同时也表明2AP探针在部分结构化的CAG环状结构域内显著堆叠，甚至可能是氢键键合。我们的研究结果表征了（CAG）（n）三联体重复DNA内的位置依赖性和构象依赖性动力学和顺序，这些结构域可能导致疾病状态的生物学机制相关的属性。
The triplet repeat sequence (CAG)(n) and related triplet repeats are associated with dynamic DNA mutations implicated in a number of debilitating human diseases. To gain insight into the dynamics of the (CAG)(n) repeat, we have substituted a single 2-aminopurine (2AP) fluorescent base for adenine at select positions within the 18 base looped domain of a (GC)(3)(CAG)(6)(GC)(3) hairpin oligonucleotide. Using temperature-dependent steady-state fluorescence measurements in combination with time correlated photon counting spectroscopy, we show the conformation and dynamics of the C2APG domains to be strongly dependent on the position of the probe in the looped region. In other words, rather than being a uniform, single stranded loop, the (CAG)6 triplet repeat looped domain exhibits order and dynamics that are position dependent. The 2AP fluorescence dynamics within the C2APG repeat are well described by a 4 component exponential decay model, with lifetimes ranging from 5 ps to 4 ns. Differences in global DNA conformation (duplex, hairpin, single strand), as well as the local position of the probe within the loop of a given hairpin, predominantly are reflected in the relative amplitude rather than the lifetime of the probe. The time dependent 2AP anisotropy in the hairpin (CAG)(n) loops is sensitive to the position of the fluorescent base, with the fluorescence depolarization of a centrally located 2AP probe within the loop proceeding significantly more slowly than 2AP positioned at the 5'- or 3'-end of the repeat sequence near the loop-stem junction. These results are consistent with segmental motions of the CAG repeat, while also suggesting that the 2AP probe is significantly stacked, possibly even hydrogen bonded, within the partially structured CAG looped domain. Our results characterize the position-dependent and conformation-dependent dynamics and order within (CAG)(n) triplet repeat DNAs, properties of relevance to the biological mechanisms by which such domains can lead to disease states.