Single-molecule analysis of DNA-binding proteins from nuclear extracts (SMADNE).
Single-molecule analysis of DNA-binding proteins from nuclear extracts (SMADNE).
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DOI：
10.1093/nar/gkad095
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发表时间：
2023-04-24
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
--
作者：
蛋白质-DNA动力学的单分子表征提供了有关许多核过程的前所未有的机械细节。在这里，我们描述了一种新的方法，快速产生单分子信息与荧光标记的蛋白质分离的人类细胞核提取物。我们使用7种天然DNA修复蛋白和2种结构变体，包括：聚（ADP-核糖）聚合酶（PARP 1），异二聚体紫外线损伤的DNA结合蛋白（UV-DDB）和8-氧代鸟嘌呤糖基化酶1（OGG 1），证明了这种新技术对未损伤DNA和3种形式的DNA损伤的广泛适用性。我们发现PARP 1与DNA缺口的结合被张力改变，并且UV-DDB在UV照射的DNA上不作为DDB 1和DDB 2的专性异源二聚体。UV-DDB与UV光产物结合的平均寿命为39秒（经光漂白校正，τc），而与8-oxoG加合物的结合寿命< 1秒。无催化活性的OGG 1变体K249 Q结合氧化损伤的时间比WT OGG 1长23倍，分别为47和2.0 s。通过同时测量三种荧光颜色，我们还表征了UV-DDB和OGG 1复合物在DNA上的组装和拆卸动力学。因此，SMADNE技术代表了一种新的、可扩展的和通用的方法，以获得对含有生理相关核蛋白的环境中的关键蛋白质-DNA相互作用的单分子机制见解。
Single-molecule characterization of protein–DNA dynamics provides unprecedented mechanistic details about numerous nuclear processes. Here, we describe a new method that rapidly generates single-molecule information with fluorescently tagged proteins isolated from nuclear extracts of human cells. We demonstrated the wide applicability of this novel technique on undamaged DNA and three forms of DNA damage using seven native DNA repair proteins and two structural variants, including: poly(ADP-ribose) polymerase (PARP1), heterodimeric ultraviolet-damaged DNA-binding protein (UV-DDB), and 8-oxoguanine glycosylase 1 (OGG1). We found that PARP1 binding to DNA nicks is altered by tension, and that UV-DDB did not act as an obligate heterodimer of DDB1 and DDB2 on UV-irradiated DNA. UV-DDB bound to UV photoproducts with an average lifetime of 39 seconds (corrected for photobleaching, τc), whereas binding lifetimes to 8-oxoG adducts were < 1 second. Catalytically inactive OGG1 variant K249Q bound oxidative damage 23-fold longer than WT OGG1, at 47 and 2.0 s, respectively. By measuring three fluorescent colors simultaneously, we also characterized the assembly and disassembly kinetics of UV-DDB and OGG1 complexes on DNA. Hence, the SMADNE technique represents a novel, scalable, and universal method to obtain single-molecule mechanistic insights into key protein–DNA interactions in an environment containing physiologically-relevant nuclear proteins.
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DOI：
10.1007/978-1-0716-0278-2_3
发表时间：
2020-01-01
期刊：
RNA SPECTROSCOPY: METHODS AND PROTOCOLS
影响因子：
--
作者：
Haraszti, Reka A.;Braun, Joerg E.
通讯作者：
Braun, Joerg E.
影响因子：
3.7
作者：
Gibb B;Ye LF;Gergoudis SC;Kwon Y;Niu H;Sung P;Greene EC
通讯作者：
Greene EC
影响因子：
56.9
作者：
Elf, Johan;Li, Gene-Wei;Xie, X. Sunney
通讯作者：
Xie, X. Sunney
DOI：
10.1126/science.1198830
发表时间：
2011-03-11
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Hoskins AA;Friedman LJ;Gallagher SS;Crawford DJ;Anderson EG;Wombacher R;Ramirez N;Cornish VW;Gelles J;Moore MJ
通讯作者：
Moore MJ
影响因子：
64.5
作者：
Ha T
通讯作者：
Ha T