Baral Saroj, Chakraborty Sagnik, Steinbach Peter J, Paul Debamita, Min Jung-Hyun, Ansari Anjum
Department of Physics, 845 W Taylor St, University of Illinois Chicago, Chicago, IL 60607, USA.
Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, NIHBC 31 BG RM 3B-62, 31 Center Drive, National Institutes of Health, Bethesda, MD 20892, USA.
Nucleic Acids Res. 2025 Jan 11;53(2). doi: 10.1093/nar/gkae1290.
Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins sense damage within genomic DNA. Using laser temperature-jump (T-jump) spectroscopy combined with cytosine-analog Förster Resonance Energy Transfer (FRET) probes that sense local DNA conformations, we measured the intrinsic dynamics of DNA containing 3 base-pair mismatches recognized in vitro by Rad4 (yeast ortholog of XPC). Rad4/XPC recognizes diverse lesions from environmental mutagens and initiates nucleotide excision repair. T-jump measurements, together with a novel and rigorous comparison with equilibrium FRET, uncovered conformational dynamics spanning multiple timescales and revealed key differences between Rad4-specific and non-specific DNA. AT-rich non-specific sites (matched or mismatched) exhibited dynamics primarily within the T-jump observation window, albeit with some amplitude in 'missing' fast (<20 μs) kinetics. These fast-kinetics amplitudes were dramatically larger for specific sites (CCC/CCC and TTT/TTT), which also exhibited 'missing' slow (>50 ms) kinetics at elevated temperatures, unseen in non-specific sites. We posit that the rapid (μs-ms) intrinsic DNA fluctuations help stall a diffusing protein at AT-rich/damaged sites and that the >50-ms kinetics in specific DNA reflect a propensity to adopt unwound/bent conformations resembling Rad4-bound DNA structures. These studies provide compelling evidence for sequence/structure-dependent intrinsic DNA dynamics and deformability that likely govern damage sensing by Rad4.
损伤位点处DNA动力学的改变与DNA修复蛋白如何感知基因组DNA中的损伤有关。我们使用激光温度跳跃（T跳跃）光谱技术，结合可感知局部DNA构象的胞嘧啶类似物荧光共振能量转移（FRET）探针，测量了含有体外被Rad4（XPC的酵母同源物）识别的3个碱基对错配的DNA的固有动力学。Rad4/XPC识别来自环境诱变剂的多种损伤并启动核苷酸切除修复。T跳跃测量，以及与平衡FRET的新颖而严格的比较，揭示了跨越多个时间尺度的构象动力学，并揭示了Rad4特异性和非特异性DNA之间的关键差异。富含AT的非特异性位点（匹配或错配）的动力学主要在T跳跃观察窗口内，尽管在“缺失”的快速（<20微秒）动力学中有一些幅度。对于特定位点（CCC/CCC和TTT/TTT），这些快速动力学幅度显著更大，在高温下，这些位点还表现出“缺失”的缓慢（>50毫秒）动力学，这在非特定位点中未见。我们认为，快速（微秒-毫秒）的固有DNA波动有助于使扩散的蛋白质在富含AT/损伤的位点停滞，并且特定位点中大于50毫秒的动力学反映了采用类似于Rad4结合DNA结构的解旋/弯曲构象的倾向。这些研究为序列/结构依赖性的固有DNA动力学和可变形性提供了有力证据，这些动力学和可变形性可能控制Rad4对损伤的感知。
