Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, NO-0372, Oslo, Norway.
Department of Microbiology, Oslo University Hospital HF, Rikshospitalet and University of Oslo, PO Box 4950 Nydalen, NO-0424, Oslo, Norway.
Nat Commun. 2018 Dec 19;9(1):5381. doi: 10.1038/s41467-018-07797-4.
In order to preserve genomic stability, cells rely on various repair pathways for removing DNA damage. The mechanisms how enzymes scan DNA and recognize their target sites are incompletely understood. Here, by using high-localization precision microscopy along with 133 Hz high sampling rate, we have recorded EndoV and OGG1 interacting with 12-kbp elongated λ-DNA in an optical trap. EndoV switches between three distinct scanning modes, each with a clear range of activation energy barriers. These results concur with average diffusion rate and occupancy of states determined by a hidden Markov model, allowing us to infer that EndoV confinement occurs when the intercalating wedge motif is involved in rigorous probing of the DNA, while highly mobile EndoV may disengage from a strictly 1D helical diffusion mode and hop along the DNA. This makes EndoV the first example of a monomeric, single-conformation and single-binding-site protein demonstrating the ability to switch between three scanning modes.
为了保持基因组的稳定性，细胞依赖于各种修复途径来清除 DNA 损伤。然而，酶如何扫描 DNA 并识别其靶位点的机制尚不完全清楚。在这里，我们通过使用高定位精度显微镜和 133Hz 的高采样率，在光阱中记录了 EndoV 和 OGG1 与 12-kbp 长 λ-DNA 的相互作用。EndoV 在三种不同的扫描模式之间切换，每种模式都有明确的激活能垒范围。这些结果与平均扩散率和隐藏 Markov 模型确定的状态占有率一致，使我们能够推断出，当嵌入楔 motif 参与到对 DNA 的严格探测中时，EndoV 会被限制，而高度移动的 EndoV 可能会脱离严格的 1D 螺旋扩散模式，并沿着 DNA 跳跃。这使得 EndoV 成为第一个能够在三种扫描模式之间切换的单体、单构象和单结合位点蛋白的例子。
