Pindi Chinmai, Ahsan Mohd, Sinha Souvik, Palermo Giulia
Department of Bioengineering, University of California Riverside, 900 University Avenue, Riverside, CA 52512, United States.
Department of Chemistry, University of California Riverside, 900 University Avenue, Riverside, CA 52512, United States.
bioRxiv. 2025 Jun 17:2025.06.17.659969. doi: 10.1101/2025.06.17.659969.
CRISPR-associated transposons (CAST) enable programmable, RNA-guided DNA integration, marking a transformative advancement in genome engineering. A central player in the type V-K CAST system is the AAA+ ATPase TnsC, which assembles into helical filaments on double-stranded DNA (dsDNA) to orchestrate target site recognition and transposition. Despite its essential role, the molecular mechanisms underlying TnsC filament nucleation and elongation remain poorly understood. Here, multiple-microsecond and free energy simulations are combined with deep learning-based Graph Attention Network (GAT) models to elucidate the mechanistic principles of TnsC filament formation and growth. Our findings reveal that ATP binding promotes TnsC nucleation by inducing DNA remodelling and stabilizing key protein-DNA interactions, particularly through conserved residues in the initiator-specific motif (ISM). Furthermore, GNN-based attention analyses identify a directional bias in filament elongation in the 5'→3' direction and uncover a dynamic compensation mechanism between incoming and bound monomers that facilitate directional growth along dsDNA. By leveraging deep learning-based graph representations, our GAT model provides interpretable mechanistic insights from complex molecular simulations and is readily adaptable to a wide range of biological systems. Altogether, these findings establish a mechanistic framework for TnsC filament dynamics and directional elongation, advancing the rational design of CAST systems with enhanced precision and efficiency.
CRISPR相关转座子（CAST）实现了可编程的、RNA引导的DNA整合，标志着基因组工程领域的一项变革性进展。V-K型CAST系统的核心元件是AAA+ATP酶TnsC，它在双链DNA（dsDNA）上组装成螺旋丝，以协调靶位点识别和转座。尽管TnsC起着至关重要的作用，但其丝状体成核和延伸的分子机制仍知之甚少。在这里，将多微秒和自由能模拟与基于深度学习的图注意力网络（GAT）模型相结合，以阐明TnsC丝状体形成和生长的机制原理。我们的研究结果表明，ATP结合通过诱导DNA重塑和稳定关键的蛋白质-DNA相互作用来促进TnsC成核，特别是通过起始子特异性基序（ISM）中的保守残基。此外，基于GNN的注意力分析确定了丝状体在5'→3'方向延伸的方向偏差，并揭示了进入的单体和结合的单体之间的动态补偿机制，这有助于沿dsDNA进行定向生长。通过利用基于深度学习的图表示，我们的GAT模型从复杂的分子模拟中提供了可解释的机制见解，并且很容易适用于广泛的生物系统。总之，这些发现建立了TnsC丝状体动力学和定向延伸的机制框架，推动了具有更高精度和效率的CAST系统的合理设计。
