Target search and recognition mechanisms of glycosylase AlkD revealed by scanning FRET-FCS and Markov state models
Target search and recognition mechanisms of glycosylase AlkD revealed by scanning FRET-FCS and Markov state models
复制标题
通过扫描 FRET-FCS 和马尔可夫态模型揭示糖基化酶 AlkD 的目标搜索和识别机制。
DOI：
10.1073/pnas.2002971117
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发表时间：
2020-09-08
影响因子：
11.1
通讯作者：
Chen，Chunlai
中科院分区：
文献类型：
--
作者：
Peng，Sijia;Wang，Xiaowei;Chen，Chunlai
DNA糖基化酶负责修复DNA损伤，维持基因组的稳定性和完整性。然而，糖基化酶如何能够有效和准确地识别巨大的DNA基因组中的DNA损伤仍然是一个谜。假设糖基化酶在寻找DNA损伤时通过在快速但低精度的扩散模式和缓慢但高精度的模式之间交替而沿着DNA易位。然而，由于目前实验技术的空间和时间分辨率的限制，慢模式还没有被成功地表征。利用新开发的扫描荧光共振能量转移（FRET）-荧光相关光谱（FCS）平台，我们能够观察到糖基化酶AlkD在双链DNA（dsDNA）上的慢和快两种模式的移位，达到微秒级的时间分辨率和亚纳米级的空间分辨率。通过全原子分子动力学模拟建立的马尔可夫态模型进一步阐明了慢模的分子机制。我们发现，在慢模式下，AlkD遵循不对称扩散途径，即，旋转，然后平移。此外，Y27在AlkD扩散动力学中的重要作用被实验和计算确定。我们的研究结果提供了机制的见解AlkD-dsDNA复合物的构象动力学如何协调不同的扩散模式，以实现高效率和准确性的DNA损伤的搜索。我们预计AlkD寻找DNA损伤的机制可能是其他糖基化酶和DNA结合蛋白所利用的一般机制。
DNA glycosylase is responsible for repairing DNA damage to maintain the genome stability and integrity. However, how glycosylase can efficiently and accurately recognize DNA lesions across the enormous DNA genome remains elusive. It has been hypothesized that glycosylase translocates along the DNA by alternating between a fast but low-accuracy diffusion mode and a slow but high-accuracy mode when searching for DNA lesions. However, the slow mode has not been successfully characterized due to the limitation in the spatial and temporal resolutions of current experimental techniques. Using a newly developed scanning fluorescence resonance energy transfer (FRET)–fluorescence correlation spectroscopy (FCS) platform, we were able to observe both slow and fast modes of glycosylase AlkD translocating on double-stranded DNA (dsDNA), reaching the temporal resolution of microsecond and spatial resolution of subnanometer. The underlying molecular mechanism of the slow mode was further elucidated by Markov state model built from extensive all-atom molecular dynamics simulations. We found that in the slow mode, AlkD follows an asymmetric diffusion pathway, i.e., rotation followed by translation. Furthermore, the essential role of Y27 in AlkD diffusion dynamics was identified both experimentally and computationally. Our results provided mechanistic insights on how conformational dynamics of AlkD–dsDNA complex coordinate different diffusion modes to accomplish the search for DNA lesions with high efficiency and accuracy. We anticipate that the mechanism adopted by AlkD to search for DNA lesions could be a general one utilized by other glycosylases and DNA binding proteins.