Fluorescence detection of cellular nucleotide excision repair of damaged DNA.
Fluorescence detection of cellular nucleotide excision repair of damaged DNA.
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DOI：
10.1038/srep05578
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发表时间：
2014-07-04
影响因子：
4.6
通讯作者：
Iwai S
中科院分区：
文献类型：
--
作者：
Toga T;Kuraoka I;Watanabe S;Nakano E;Takeuchi S;Nishigori C;Sugasawa K;Iwai S
为了保持遗传完整性，DNA中紫外光诱导的光产物必须通过核苷酸切除修复（NER）途径去除，这是由损伤识别和含损伤链的双切口启动的。我们打算用荧光探针检测细胞内隐核的双切口步骤。首先制备了含有（6-4）光产物和荧光团猝灭剂对的140碱基对线性双相。然而，这种类型的DNA被发现在细胞中被核酸酶迅速降解。接下来，一个质粒被用作支架。在这种情况下，荧光团和猝灭剂附着在同一条链上，我们预计含有它们的双切口产物将在细胞中降解。质粒型探针转染HeLa细胞3 h后，只有在探针含有（6-4）光产物时，荧光显微镜才能检测到细胞核的荧光发射，并通过流式细胞术证实了这一结果。最后，用含光产物探针转染XPA成纤维细胞和表达XPA基因的相同细胞。虽然探针进入细胞的转移速度较慢，但根据细胞的NER能力可以检测到荧光。
To maintain genetic integrity, ultraviolet light-induced photoproducts in DNA must be removed by the nucleotide excision repair (NER) pathway, which is initiated by damage recognition and dual incisions of the lesion-containing strand. We intended to detect the dual-incision step of cellular NER, by using a fluorescent probe. A 140-base pair linear duplex containing the (6–4) photoproduct and a fluorophore–quencher pair was prepared first. However, this type of DNA was found to be degraded rapidly by nucleases in cells. Next, a plasmid was used as a scaffold. In this case, the fluorophore and the quencher were attached to the same strand, and we expected that the dual-incision product containing them would be degraded in cells. At 3 h after transfection of HeLa cells with the plasmid-type probes, fluorescence emission was detected at the nuclei by fluorescence microscopy only when the probe contained the (6–4) photoproduct, and the results were confirmed by flow cytometry. Finally, XPA fibroblasts and the same cells expressing the XPA gene were transfected with the photoproduct-containing probe. Although the transfer of the probe into the cells was slow, fluorescence was detected depending on the NER ability of the cells.
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影响因子：
5.3
作者：
Otrin, VR;Kuraoka, I;Wood, RD
通讯作者：
Wood, RD
影响因子：
14.9
作者：
Limsirichaikul S;Niimi A;Fawcett H;Lehmann A;Yamashita S;Ogi T
通讯作者：
Ogi T
影响因子：
4.8
作者：
MATSUNAGA, T;MU, D;SANCAR, A
通讯作者：
SANCAR, A
影响因子：
15
作者：
Iwai, S;Shimizu, M;Ohtsuka, E
通讯作者：
Ohtsuka, E
影响因子：
4
作者：
Bradford PT;Goldstein AM;Tamura D;Khan SG;Ueda T;Boyle J;Oh KS;Imoto K;Inui H;Moriwaki S;Emmert S;Pike KM;Raziuddin A;Plona TM;DiGiovanna JJ;Tucker MA;Kraemer KH
通讯作者：
Kraemer KH