Transcription arrest at a lesion in the transcribed DNA strand in vitro is not affected by a nearby lesion in the opposite strand
Transcription arrest at a lesion in the transcribed DNA strand in vitro is not affected by a nearby lesion in the opposite strand
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DOI：
10.1074/jbc.m301060200
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发表时间：
2003-05-23
影响因子：
4.8
通讯作者：
Hanawalt, PC
中科院分区：
文献类型：
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作者：
Kalogeraki, VS;Tornaletti, S;Hanawalt, PC
顺式环丁烷嘧啶二聚体 (CPD) 是紫外线照射 DNA 中最常形成的损伤。 CPD 通过核苷酸切除修复途径进行修复。此外，它们还受到转录偶联 DNA 修复的影响。在转录偶联 DNA 修复的一般模型中，在转录 DNA 链上的损伤处停滞的 RNA 聚合酶通过将修复机制募集到损伤部位来促进修复。与该模型一致，体外转录实验表明，转录DNA链中的CPD会干扰原核和真核RNA聚合酶的易位。在这里，我们研究了 RNA 聚合酶在转录包含两个紧密间隔的损伤（每条 DNA 链上一个）的模板时的行为。使用不含 CPD 或转录或非转录链上单个 CPD 的类似 DNA 模板作为对照。使用具有纯化的 T7 RNA 聚合酶 (T7 RNAP) 或大鼠肝脏 RNAP II 的体外转录系统，我们表征了体外转录的长度和效率。我们还测试了转录链 CPD 处停滞的 RNAP II-DNA-RNA 三元复合物对转录因子 TFIIS 的敏感性。非转录链中附近 CPD 的存在不会影响任一 RNA 聚合酶的行为，也不会影响 RNAP II 停滞复合物的反向易位能力。我们的结果还表明，CPD 的序列背景对 T7 RNAP 阻滞效率的影响比 RNAP II 更显着。
Cis-syn cyclobutane pyrimidine dimers (CPDs) are the most frequently formed lesions in UV-irradiated DNA. CPDs are repaired by the nucleotide excision repair pathway. Additionally, they are subject to transcription-coupled DNA repair. In the general model for transcription-coupled DNA repair, an RNA polymerase arrested at a lesion on the transcribed DNA strand facilitates repair by recruiting the repair machinery to the site of the lesion. Consistent with this model, transcription experiments in vitro have shown that CPDs in the transcribed DNA strand interfere with the translocation of prokaryotic and eukaryotic RNA polymerases. Here, we study the behavior of RNA polymerase when transcribing a template that contains two closely spaced lesions, one on each DNA strand. Similar DNA templates containing no CPD, or a single CPD on either the transcribed or the nontranscribed strand were used as controls. Using an in vitro transcription system with purified T7 RNA polymerase ( T7 RNAP) or rat liver RNAP II, we characterized transcript length and efficiency of transcription in vitro. We also tested the sensitivity of the arrested RNAP II-DNA-RNA ternary complex, at a CPD in the transcribed strand, to transcription factor TFIIS. The presence of a nearby CPD in the nontranscribed strand did not affect the behavior of either RNA polymerase nor did it affect the reverse translocation ability of the RNAP II-arrested complex. Our results additionally indicate that the sequence context of a CPD affects the efficiency of T7 RNAP arrest more significantly than that of RNAP II.