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同源重组是维持基因组稳定和生物多样性的核心机制.RecA作为最早被发现的同源重组酶，在同源重组链交换过程中起着关键作用.关于同源重组机制的研究已持续数十年，近年来使用单分子技术使得该领域研究取得了不少重大突破.其中单分子FRET（ Fluorescence Resonance EnergyTransfer）技术的应用最为广泛，然而FRET实验所必需的荧光标记可能会对RecA介导的链交换过程产生影响，且往往会被研究人员所忽视.本文通过对不同标记位置和标记方式进行梳理，将荧光标记对同源重组链交换的影响总结为链特异性和构象敏感性，并给出了影响最小的标记方案.该结果增加了对荧光标记影响的理解，研究人员可以快速优化荧光标记位置和方式，降低其对链交换过程带来的负面影响，也对其他荧光标记实验有一定的启发意义.Homologous recombination is a central mechanism for maintaining genome stability and biodiversity. RecA, as the first discovered homologous recombinase, plays a crucial role in homologous recombination strand exchange. In recent years, with the development of structural biology, significant breakthroughs have been made in understanding the static structure of the RecA nucleoprotein filament. However, research on the kinetic process of homologous recombination strand exchange mediated by RecA continues to face enormous challenges. Research on the dynamic process has been ongoing for decades. In recent years, the application of single-molecule techniques has led to significant breakthroughs in this field. Among these techniques, single-molecule fluorescence resonance energy transfer (FRET) technology is widely used due to its ultra-high temporal and spatial resolution, making it well-suited for studying RecA-mediated homologous recombination strand exchange. However, the fluorescent labels required for FRET experiments may affect the RecA-mediated strand exchange process, which is often overlooked by researchers. Most related articles focus on the impact of fluorescent labels on local structure. This paper primarily explores the impact of DNA fluorescent labeling on protein function, and understands its influence on strand exchange from two perspectives: the strand specificity and conformational sensitivity of fluorescent labeling. Through experiments such as double strand binding, single strand invasion, and strand exchange, we obtained a labeling scheme with the minimal impact—9 bp spaced Cstrand double base labeling in triplet, which can effectively improve the efficiency of studying the homologous recombination process. This result enhances the understanding of the impact of fluorescent labeling, allowing researchers to rapidly optimize the position and method of fluorescent labeling, and reduce its negative effects on the strand exchange process. Moreover, it provides some inspiration for other fluorescent labeling experiments.
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Keywords:
- Homologous recombination /
- RecA /
- Single-molecule FRET /
- Fluorescent labeling
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[1] Kowalczykowski S C, Dixon D A, Eggleston A K, Lauder S D, Rehrauer W M 1994 Microbiol. Rev. 58 401
[2] Prentiss M, Prévost C, Danilowicz C 2015 Crit. Rev. Biochem. Mol. Biol. 50 453
[3] Roca A I, Cox M M 1990 Crit. Rev. Biochem. Mol. Biol. 25 415
[4] West S C, Howard-Flanders P 1984 Cell 37 683
[5] Yang H, Zhou C, Dhar A, Pavletich N P 2020 Nature 586 801
[6] Danilowicz C, Feinstein E, Conover A, Coljee V W, Vlassakis J, Chan Y-L, Bishop D K, Prentiss M 2012 Nucleic Acids Res. 40 1717
[7] Mäeots M E, Lee B, Nans A, Jeong S G, Esfahani M M N, Ding S, Smith D J, Lee C S, Lee S S, Peter M, Enchev R I 2020 Nat. Commun. 11 3465
[8] Hertzog M, Perry T N, Dupaigne P, Serres S, Morales V, Soulet A L, Bell J C, Margeat E, Kowalczykowski S C, Le Cam E, Fronzes R, Polard P 2023 Nucleic Acids Res. 51 2800
[9] Chen J, Tang Q, Guo S, Lu C, Le S, Yan J 2017 Nucleic Acids Res. 45 10032
[10] Axelrod D 1984 J. Lumin. 31–32 881
[11] Groves D, Hepp C, Kapanidis A N, Robb N C 2023 Quart. Rev. Biophys. 56 e3
[12] Kim S H, Ragunathan K, Park J, Joo C, Kim D, Ha T 2014 J. Am. Chem. Soc. 136 14796
[13] Ragunathan K, Liu C, Ha T 2012 eLife 1 e00067
[14] Yu F, Zhang D, Zhao C, Zhao Q, Jiang G, Wang H 2023 Nucleic Acids Res. 51 2270
[15] Kim S H, Joo C, Ha T, Kim D 2013 Nucleic Acids Res. 41 7738
[16] Joo C, McKinney S A, Nakamura M, Rasnik I, Myong S, Ha T 2006 Cell 126 515
[17] Garavís M, Escaja N, Gabelica V, Villasante A, González C 2015 Chem. Eur. J. 21 9816
[18] Cox J M, Tsodikov O V, Cox M M 2005 PLoS Biol. 3 e52
[19] Huang X Y, Sui M Y, Hou W Q, Li M, Lu Y, Xu C H, 2020 Acta Phys. Sin. 69 208706 (黄星榞, 隋明宇, 侯文清, 李明, 陆颖, 徐春华 2020 69 208706)
[20] Roy R, Hohng S, Ha T 2008 Nat. Methods 5 507
[21] Peacock-Villada A, Yang D, Danilowicz C, Feinstein E, Pollock N, McShan S, Coljee V, Prentiss M 2012 Nucleic Acids Res. 40 10441
[22] Huang X, Lu Y, Wang S, Sui M, Li J, Ma J, Ma D, Jia Q, Hu S, Xu C, Li M 2020 Proc. Natl. Acad. Sci. U.S.A. 117 20549
[23] Zasedateleva O A, Vasiliskov V A, Surzhikov S A, Kuznetsova V E, Shershov V E, Guseinov T O, Smirnov I P, Yurasov R A, Spitsyn M A, Chudinov A V 2018 Nucleic Acids Res. 46 e73
[24] Chen Z, Yang H, Pavletich N P 2008 Nature 453 489
[25] Heussman D, Kittell J, Von Hippel P H, Marcus A H 2022 J. Chem. Phys. 156 045101
[26] Jahnke K, Grubmüller H, Igaev M, Göpfrich K 2021 Nucleic Acids Res. 49 4186
[27] Cutler J I, Zhang K, Zheng D, Auyeung E, Prigodich A E, Mirkin C A 2011 J. Am. Chem. Soc. 133 9254
[28] Cavaco M, Pérez-Peinado C, Valle J, Silva R D M, Correia J D G, Andreu D, Castanho M A R B, Neves V 2020 Front. Bioeng. Biotechnol. 8 552035
[29] Yin L, Wang W, Wang S, Zhang F, Zhang S, Tao N 2015 Biosens. Bioelectron. 66 412
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