Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand.
Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand.
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DOI：
10.1093/nar/gkr704
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发表时间：
2012-01
影响因子：
14.9
通讯作者：
Queimado L
中科院分区：
文献类型：
--
作者：
Reis AM;Mills WK;Ramachandran I;Friedberg EC;Thompson D;Queimado L
内源性DNA损伤主要通过碱基切除修复（BER）来消除，然而内源性DNA损伤是否存在优先链修复仍存在激烈的争论。我们开发了一种高灵敏度的引物锚定DNA损伤检测方法（PADDA），以定位和定量体内内源性DNA损伤。使用PADDA，我们记录了显着更高水平的内源性损伤酿酒酵母细胞在稳定期比在指数期。我们还证明了酵母BER缺陷细胞在生长的任何阶段都比同基因野生型细胞具有显著更高水平的内源性DNA损伤。PADDA在单核苷酸水平上提供了详细的指纹分析，首次记录了CAN1中持续的内源性核苷酸损伤与先前报道的自发CAN1突变共定位。为了快速可靠地定量组成型表达的CAN1基因中的内源性链特异性DNA损伤，我们在实时PCR设置中使用PADDA。我们证明，野生型细胞修复内源性损伤优先对CAN1转录链。相比之下，酵母BER缺陷型细胞优先在CAN1转录链上积累内源性损伤。这些数据提供了第一个直接的证据，优先链修复内源性DNA损伤和文件的BER在这一过程中的主要作用。
Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.
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影响因子：
4.1
作者：
Encell, L;Shuker, DEG;Gold, B
通讯作者：
Gold, B
DOI：
10.1016/0027-5107(90)90173-2
发表时间：
1990-07-01
期刊：
MUTATION RESEARCH
影响因子：
--
作者：
BERANEK, DT
通讯作者：
BERANEK, DT
影响因子：
2.9
作者：
Drouin, R;Vigneault, F
通讯作者：
Vigneault, F
影响因子：
9.2
作者：
Bruner, SD;Nash, HM;Verdine, GL
通讯作者：
Verdine, GL
影响因子：
16
作者：
Brégeon, D;Doddridge, ZA;Doetsch, PW
通讯作者：
Doetsch, PW