Mechanism of damage recognition by Rad4/XPC in linear and circular DNA
Mechanism of damage recognition by Rad4/XPC in linear and circular DNA
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Rad4/XPC 识别线性和环状 DNA 损伤的机制
DOI：
10.1016/j.bpj.2023.11.550
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发表时间：
2024
影响因子：
3.4
通讯作者：
Ansari, Anjum
中科院分区：
文献类型：
--
作者：
Baral, Saroj;Chakraborty, Sagnik;Paul, Debamita;Pigli, Ying;Steinbach, Peter J.;Min, Jung-Hyun;Rice, Phoebe A.;Ansari, Anjum
病变部位的DNA动态变化与DNA修复蛋白如何暂停和识别基因组DNA中的损伤有关。描述这些动态一直是一项挑战。我们在Rad4(XPC的酵母同源基因)损伤识别的背景下研究了DNA动力学，它识别不同的损伤来自紫外线损伤或其他基因毒素，并启动核苷酸切除修复。以前的研究将胞嘧啶-类似FRET对放置在3个碱基对(BP)错配位点的两侧--在体外被Rad4特异性识别--揭示了即使没有Rad4也会出现严重变形的DNA(Chakraborty等人(2018年)，核酸研究46：1240-1255)。在这里，我们使用激光T跳跃，揭示了这些自发变形的时间尺度。富含AT的非特异性位点，无论匹配或错配，主要在∼20μS-<；100ms的T-跳跃观察窗口内显示构象动力学，尽管在未分辨(<；20-μS)动力学中有一些幅度。对于特定的位置，“隐藏的”快速动力学的幅度显著增加，这进一步被T-JUMP观察不到的太慢(>；100-ms)的动力学的额外幅度所区别。我们假设<；20-μS的波动有助于阻止扩散的蛋白质在AT丰富/受损的部位，并且>；100-ms动力学反映了特定DNA采用可能类似Rad4结合结构的展开/弯曲构象的倾向。这些研究为Rad4可以感觉到的不寻常的DNA动力学和变形性提供了令人信服的证据。上述研究是用24bp线性DNA寡聚体进行的，并不能反映环状和/或超螺旋DNA的构象动力学。为了检验循环的影响，我们在扭转松弛的126-bp DNA小环中加入了一个错配的位置，并且与线性DNA相比，未覆盖的>；Rad4结合亲和力增加了100倍。这些结果表明，关于循环和超级卷曲如何影响DNA损伤感知，还有很多有待发现的地方。
Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins pause and identify damage within genomic DNA. Characterizing these dynamics has been a challenge. We examined DNA dynamics in the context of damage recognition by Rad4 (yeast ortholog of XPC), which recognizes diverse lesions from UV-damage or other genotoxins and initiates nucleotide excision repair. Previous studies with a cytosine-analog FRET pair placed on either side of 3 base-pair (bp) mismatched sites—recognized specifically by Rad4 in vitro—unveiled severely deformed DNA even without Rad4 (Chakraborty et al.(2018), Nucleic Acid Res. 46: 1240–1255). Here, using laser T-jump, we revealed the timescales of these spontaneous deformations. Nonspecific sites that were AT-rich, whether matched or mismatched, exhibited conformational dynamics primarily within the T-jump observation window of∼ 20 μs-< 100 ms, albeit with some amplitude in unresolved (< 20-μs) kinetics. The amplitudes of the “hidden” fast kinetics increased dramatically for specific sites, which were further distinguished by additional amplitude in kinetics that were too slow (> 100-ms) to be observed with T-jump. We posit that the< 20-μs fluctuations help stall a diffusing protein at AT-rich/damaged sites and that the> 100-ms kinetics reflect a propensity for specific DNA to adopt unwound/bent conformations that may resemble Rad4-bound structures. These studies provide compelling evidence for unusual DNA dynamics and deformability that Rad4 can sense. The above studies, done with 24-bp linear DNA oligomers, do not reflect the conformational dynamics expected of looped and/or supercoiled DNA. To examine the impact of looping, we incorporated a mismatched site within torsionally-relaxed 126-bp DNA minicircles and uncovered> 100-fold increase in Rad4 binding affinity compared with linear DNA. These results showcase that much remains to be discovered regarding how looping and supercoiling impact DNA damage sensing.