Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair.
Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair.
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DOI：
10.1186/1471-2091-12-2
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发表时间：
2011-01-08
期刊：
影响因子：
--
通讯作者：
Zhou P
中科院分区：
文献类型：
--
作者：
An J;Yang T;Huang Y;Liu F;Sun J;Wang Y;Xu Q;Wu D;Zhou P
在真核细胞中，核苷酸切除修复（NER）有两个子途径，即全基因组（gg）NER和转录偶联修复（TCR）。TCR可以优先去除位于转录活性基因的转录链处的大体积DNA损伤，比位于未转录链或整个基因组DNA处的大体积DNA损伤更快。在合适的限制性片段中的这种链特异性修复通常通过碱性凝胶电泳，随后通过Southern印迹转移和与间接末端标记的单链探针杂交来确定。本文介绍了一种基于链特异性PCR（SS-PCR）的TCR检测新方法。使用这种方法，我们已经调查的作用，DNA依赖性蛋白激酶催化亚基（DNA-PKcs），磷脂酰肌醇3-激酶相关的蛋白激酶（PIKK）家族的成员，在紫外线诱导的DNA损伤的TCR途径。尽管DNA-PKcs的耗竭使HeLa细胞对UV辐射敏感，但它并不影响UV诱导的环丁烷嘧啶二聚体（CPD）损伤的ggNER效率。我们推测DNA-PKcs可能参与TCR过程。为了验证这一假设，我们首先开发了一种新的TCR检测方法，该方法基于一组智能引物的链特异性PCR技术，该技术允许对哺乳动物细胞中UV辐射损伤的基因组DNA的限制性基因片段进行链特异性扩增。使用这种新方法，我们证实了siRNA介导的Cockayne综合征B下调导致了紫外线损伤的二氢叶酸还原酶（DHFR）基因TCR的缺陷。另外，DMSO诱导的c-myc基因沉默导致HL 60细胞中UV辐射损伤的c-myc基因的TCR效率降低。在上述方法学验证的基础上，我们发现siRNA介导的DNA-PKcs缺失显著降低了HeLa细胞DHFR基因中TCR修复UV诱导的CPD损伤的能力，表明DNA-PKcs可能也参与了DNA损伤修复的TCR途径。通过免疫沉淀和MALDI-TOF-质谱分析，我们揭示了DNA-PKcs和细胞周期蛋白T2的相互作用，这是一个亚基的人转录延伸因子（P-TEFb）。而P-TEFb复合物可以磷酸化RNA聚合酶II羧基末端结构域（CTD）的丝氨酸2，并促进转录延伸。建立了一种基于链特异性PCR（SS-PCR）的TCR检测新方法。我们的数据表明，DNA-PKcs在紫外线损伤的DNA的TCR途径中起作用。一种可能的机制假说是DNA-PKcs可能通过与CyclinT 2/CDK 9（P-TEFb）结合来调节RNA Pol II的活性，RNA Pol II已被鉴定为识别和启动TCR的关键分子。
In eukaryotic cells, there are two sub-pathways of nucleotide excision repair (NER), the global genome (gg) NER and the transcription-coupled repair (TCR). TCR can preferentially remove the bulky DNA lesions located at the transcribed strand of a transcriptional active gene more rapidly than those at the untranscribed strand or overall genomic DNA. This strand-specific repair in a suitable restriction fragment is usually determined by alkaline gel electrophoresis followed by Southern blotting transfer and hybridization with an indirect end-labeled single-stranded probe. Here we describe a new method of TCR assay based on strand-specific-PCR (SS-PCR). Using this method, we have investigated the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related protein kinases (PIKK) family, in the TCR pathway of UV-induced DNA damage. Although depletion of DNA-PKcs sensitized HeLa cells to UV radiation, it did not affect the ggNER efficiency of UV-induced cyclobutane pyrimidine dimers (CPD) damage. We postulated that DNA-PKcs may involve in the TCR process. To test this hypothesis, we have firstly developed a novel method of TCR assay based on the strand-specific PCR technology with a set of smart primers, which allows the strand-specific amplification of a restricted gene fragment of UV radiation-damaged genomic DNA in mammalian cells. Using this new method, we confirmed that siRNA-mediated downregulation of Cockayne syndrome B resulted in a deficiency of TCR of the UV-damaged dihydrofolate reductase (DHFR) gene. In addition, DMSO-induced silencing of the c-myc gene led to a decreased TCR efficiency of UV radiation-damaged c-myc gene in HL60 cells. On the basis of the above methodology verification, we found that the depletion of DNA-PKcs mediated by siRNA significantly decreased the TCR capacity of repairing the UV-induced CPDs damage in DHFR gene in HeLa cells, indicating that DNA-PKcs may also be involved in the TCR pathway of DNA damage repair. By means of immunoprecipitation and MALDI-TOF-Mass spectrometric analysis, we have revealed the interaction of DNA-PKcs and cyclin T2, which is a subunit of the human transcription elongation factor (P-TEFb). While the P-TEFb complex can phosphorylate the serine 2 of the carboxyl-terminal domain (CTD) of RNA polymerase II and promote transcription elongation. A new method of TCR assay was developed based the strand-specific-PCR (SS-PCR). Our data suggest that DNA-PKcs plays a role in the TCR pathway of UV-damaged DNA. One possible mechanistic hypothesis is that DNA-PKcs may function through associating with CyclinT2/CDK9 (P-TEFb) to modulate the activity of RNA Pol II, which has already been identified as a key molecule recognizing and initializing TCR.
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