Esadze Alexandre, Rodriguez Gaddiel, Weiser Brian P, Cole Philip A, Stivers James T
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA.
Nucleic Acids Res. 2017 Dec 1;45(21):12413-12424. doi: 10.1093/nar/gkx848.
DNA 'sliding' by human repair enzymes is considered to be important for DNA damage detection. Here, we transfected uracil-containing DNA duplexes into human cells and measured the probability that nuclear human uracil DNA glycosylase (hUNG2) excised two uracil lesions spaced 10-80 bp apart in a single encounter without escaping the micro-volume containing the target sites. The two-site transfer probabilities were 100% and 54% at a 10 and 40 bp spacing, but dropped to only 10% at 80 bp. Enzyme trapping experiments suggested that site transfers over 40 bp followed a DNA 'hopping' pathway in human cells, indicating that authentic sliding does not occur even over this short distance. The transfer probabilities were much greater than observed in aqueous buffers, but similar to in vitro measurements in the presence of polymer crowding agents. The findings reveal a new role for the crowded nuclear environment in facilitating DNA damage detection.
人类修复酶介导的DNA“滑动”被认为对DNA损伤检测很重要。在此，我们将含尿嘧啶的DNA双链体转染到人类细胞中，并测量了细胞核中的人类尿嘧啶DNA糖基化酶（hUNG2）在单次接触中切除两个相距10 - 80碱基对的尿嘧啶损伤且不逃离包含靶位点的微体积区域的概率。在10个碱基对和40个碱基对的间距下，两位点转移概率分别为100%和54%，但在80个碱基对时降至仅10%。酶捕获实验表明，在人类细胞中超过40个碱基对的位点转移遵循DNA“跳跃”途径，这表明即使在这么短的距离内也不会发生真正的滑动。转移概率远高于在水性缓冲液中观察到的结果，但与在存在聚合物拥挤剂的体外测量结果相似。这些发现揭示了拥挤的细胞核环境在促进DNA损伤检测中的新作用。
