Department of Medical Laboratory and Radiation Sciences, University of Vermont, Burlington, VT 05405, USA.
Cancer Cell Int. 2007 Sep 24;7:15. doi: 10.1186/1475-2867-7-15.
Major genomic surveillance mechanisms regulated in response to DNA damage exist at the G1/S and G2/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation.
Using an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined.
Overall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of enhanced BER activities was found in irradiated cells arrested in G2 phase.
存在针对 DNA 损伤调控的主要基因组监测机制，分别在 G1/S 和 G2/M 检查点。据推测，这些延迟为修复受损 DNA 提供了时间。细胞已经开发出多种 DNA 修复途径来保护自己免受不同类型的 DNA 损伤。氧化 DNA 损伤由碱基切除修复 (BER) 途径处理。关于电离辐射诱导的 DNA 损伤的 BER 以及细胞周期背景下 BER 的潜在异质性知之甚少。我们在整个细胞周期中测量了三种 BER 酶的活性，以研究电离辐射诱导的 DNA 损伤的细胞周期特异性修复。我们进一步研究了在暴露于电离辐射后 G2 期被阻断的人细胞中的 BER 活性。
使用涉及具有特定 DNA 损伤的放射性标记寡核苷酸的体外切口测定法，我们在从 G1 和 G2 期细胞周期中照射的同步化人 HeLa 细胞的全细胞提取物中，检查了几种 BER 酶的活性。人内切核酸酶 III (hNTH1)的活性，一种从 DNA 中去除几种受损碱基的糖苷酶/裂解酶，包括二氢尿嘧啶 (DHU)、识别 7,8-二氢-8-氧代-2'-脱氧鸟苷 (8-oxoG)损伤的 8-氧鸟嘌呤-DNA 糖苷酶 (hOGG1)和作用于包括合成类似物呋喃的无碱基位点的脱嘌呤/脱嘧啶内切核酸酶 (hAPE1)。
总体而言，hNTH1 和 hAPE1 的修复活性在细胞周期的 G1 期比 G2 期更高。在 G1 和 G2 期，具有呋喃的寡核苷酸底物的切割率大于具有 DHU 的寡核苷酸底物。仅在 G1 期，细胞照射增强了具有呋喃和 DHU 的底物的切割。hOGG1 的活性要低得多，并且在细胞周期内没有变化。这些结果表明 BER 对电离辐射诱导的 DNA 损伤具有细胞周期依赖性。有趣的是，在 G2 期被阻断的照射细胞中未发现 BER 活性增强的证据。
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