Department of Pharmacology and Toxicology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA and Department of Radiation Oncology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.
Nucleic Acids Res. 2014 Mar;42(5):3125-37. doi: 10.1093/nar/gkt1347. Epub 2013 Dec 25.
To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3'-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3'-phosphate and 3'-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3'-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3'-PG and 3'-phosphate termini on 1-base 3' overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3'-PG termini were processed more slowly than 3'-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3'-PG blocking groups for DSB repair. DSBs with 3'-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3'-PG ends.
为了追踪体内受损 DNA 双链断裂 (DSB) 末端的处理过程，设计了一种方法来定量测量人 Alu 重复序列中非蛋白生色团新制癌菌素 (NCS-C) 诱导的 DSB 上的 3'-磷酸甘油酸 (PG) 末端。在细胞暴露于 NCS-C 后，分离 DNA，并通过化学方法稳定易损损伤。所有 3'-磷酸和 3'-羟基末端均通过双脱氧末端酶促封闭，而 3'-PG 末端则可连接，连接到锚定物，并通过实时 Taqman 聚合酶链反应进行定量。使用该测定法及其变体，很容易在经处理的淋巴母细胞系 DNA 中检测到 NCS-C 诱导的 DSB 上的 1 个碱基 3'突出端的 3'-PG 和 3'-磷酸末端，并且在 1 小时内，这两种末端均从细胞 DNA 中大量消除。但是，3'-PG 末端的处理速度比 3'-磷酸末端慢，并且在酪氨酸-DNA 磷酸二酯酶 1 突变体 SCAN1 中比在正常细胞中更持久，这表明酪氨酸-DNA 磷酸二酯酶 1 在去除 DSB 修复的 3'-PG 阻断基团方面起着重要作用。带有 3'-羟基末端的 DSB，这些末端不是由 NCS-C 直接诱导的，在细胞中迅速形成，并且在 1 小时内通过进一步处理在很大程度上消除，在 Alu 重复序列中和异染色质 α-卫星 DNA 中均如此。此外，M059J 细胞中缺乏 DNA-PK 似乎加速了 3'-PG 末端的解决。
