Highly specific and sensitive method for measuring nucleotide excision repair kinetics of ultraviolet photoproducts in human cells.
Highly specific and sensitive method for measuring nucleotide excision repair kinetics of ultraviolet photoproducts in human cells.
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DOI：
10.1093/nar/gkt1179
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发表时间：
2014-02
影响因子：
14.9
通讯作者：
Sancar A
中科院分区：
文献类型：
--
作者：
Choi JH;Gaddameedhi S;Kim SY;Hu J;Kemp MG;Sancar A
核苷酸切除修复途径以长度为30个核苷酸的短寡核苷酸∼的形式从人类基因组中移除紫外线(UV)光产物。由于目前研究体内紫外光产物修复的许多方法都存在局限性，我们开发了一种方便的非放射性同位素方法来直接检测人类细胞中的DNA切除修复事件。该方法包括从紫外线照射的细胞中提取寡核苷酸，用生物素和链霉亲和素介导的DNA末端标记化学发光检测切除后的基因组在切除修复过程中释放的含有紫外光产物的寡核苷酸。我们的新方法是稳健的，在没有紫外线或功能切除修复系统的情况下基本上没有信号。此外，我们的非放射性同位素方法允许在紫外线照射后几分钟内灵敏地检测切除产物，而不需要额外的浓缩步骤，如免疫沉淀。最后，这项技术允许对人类细胞的切除修复进行定量测量。我们认为，这些新技术将是在活体内研究人类核苷酸切除修复机制的一种有用而有力的方法。
The nucleotide excision repair pathway removes ultraviolet (UV) photoproducts from the human genome in the form of short oligonucleotides ∼30 nt in length. Because there are limitations to many of the currently available methods for investigating UV photoproduct repair in vivo, we developed a convenient non-radioisotopic method to directly detect DNA excision repair events in human cells. The approach involves extraction of oligonucleotides from UV-irradiated cells, DNA end-labeling with biotin and streptavidin-mediated chemiluminescent detection of the excised UV photoproduct-containing oligonucleotides that are released from the genome during excision repair. Our novel approach is robust, with essentially no signal in the absence of UV or a functional excision repair system. Furthermore, our non-radioisotopic methodology allows for the sensitive detection of excision products within minutes following UV irradiation and does not require additional enrichment steps such as immunoprecipitation. Finally, this technique allows for quantitative measurements of excision repair in human cells. We suggest that the new techniques presented here will be a useful and powerful approach for studying the mechanism of human nucleotide excision repair in vivo.
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影响因子：
3.8
作者：
Mouret, Stephane;Charveron, Marie;Douki, Thierry
通讯作者：
Douki, Thierry
DOI：
10.1073/pnas.51.2.293
发表时间：
1964-01-01
影响因子：
11.1
作者：
BOYCE, RP;HOWARDFLANDERS, P
通讯作者：
HOWARDFLANDERS, P
DOI：
10.1073/pnas.51.2.226
发表时间：
1964-01-01
影响因子：
11.1
作者：
SETLOW, RB;CARRIER, WL
通讯作者：
CARRIER, WL
影响因子：
3.3
作者：
LABELLE, M;LINN, S
通讯作者：
LINN, S
影响因子：
3.3
作者：
Nishinaga, Mari;Kurata, Ryuichiro;Matsunaga, Tsukasa
通讯作者：
Matsunaga, Tsukasa