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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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