Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions
Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions
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DOI：
10.1073/pnas.1415186112
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发表时间：
2015-02-03
影响因子：
11.1
通讯作者：
Kashlev, Mikhail
中科院分区：
文献类型：
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作者：
Walmacq, Celine;Wang, Lanfeng;Kashlev, Mikhail
在人类细胞中，氧化性DNA损伤8，5 '-环-2'-脱氧腺苷（CydA）诱导RNA聚合酶II（Pol II）的长时间停滞，随后是转录旁路，产生无错误和突变的转录物，其中AMP错误地掺入损伤的下游。在这里，我们提出的生化和晶体学证据的机制CydA识别。Pol II停滞是由于CydA旁边的模板碱基（5 '）加载到活性位点中受损导致优先AMP错误掺入。这种主要的AMP插入，也发生在脱碱基位点，不受5 '-模板碱基身份的影响，表明它来源于非模板合成，根据DNA聚合酶已知的A规则，最近被鉴定为嘧啶二聚体的Pol II旁路。在AMP错误掺入之后，Pol II遇到了一个主要的易位阻滞，该阻滞被缓慢克服。因此，易位阻断与dA.rA错配的不良延伸相结合减少了转录突变。此外，通过触发环中的突变来增加活性位点的灵活性，这增加了Pol II适应大体积病变的能力，并且添加反式作用因子TFIIF促进CydA旁路。因此，阻断损伤进入活性位点、跨损伤A规则合成和易位阻断是跨不同大体积DNA损伤的转录的共同特征。
In human cells, the oxidative DNA lesion 8,5 '-cyclo-2 '-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5 ') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5 '-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.