The Nonbulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in Vitro.
The Nonbulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in Vitro.
复制标题
DOI：
10.1021/acs.biochem.7b00295
复制
发表时间：
2017-06-20
期刊：
影响因子：
2.9
通讯作者：
Shafirovich V
中科院分区：
文献类型：
--
作者：
Kolbanovskiy M;Chowdhury MA;Nadkarni A;Broyde S;Geacintov NE;Scicchitano DA;Shafirovich V
细胞DNA中最常见的氧化损伤是8-氧代-7，8-二氢鸟嘌呤，它可以进一步氧化，在DNA中产生高度诱变的螺亚氨基二海因(Sp)和5-胍基海因(Gh)。在人类无细胞提取物中，这两种病变都可以通过碱基切除修复和全基因组核苷酸切除修复来切除。然而，目前尚不清楚这些损伤是否可以通过转录偶联DNA修复(TCR)来移除，TCR是一种从阻碍RNA合成的DNA中清除损伤的途径。为了确定Sp或Gh是否阻碍转录，使它们成为TCR的有效底物，在支持人RNA聚合酶II转录的启动子的控制下，将Sp或Gh损伤定位在DNA转录链上。这些构建体在含有活性RNA聚合酶II的HeLa核提取液中孵育，所得转录物通过变性聚丙烯酰胺凝胶电泳进行解析。结构刚性的Sp强烈阻止转录延伸，允许名义损伤旁路1.6±0.5%。相反，构象柔性的Gh对人RNAPII的阻碍较小，允许9±2%的旁路。此外，Sp和Gh的部分病变搭桥术受HeLa核提取液中发现的糖基酶活性的影响最小。这些数据特别表明，Sp和Gh都很可能对TCR易感，因为它们都对人类RNA聚合酶II的进展构成了显著的阻碍。还提出了一个更一般的原则：构象灵活性可能是DNA损伤的一个重要结构特征，它增强了它们的转录旁路。
The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impede transcription, which could make them viable substrates for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting nominal lesion bypass of 1.6 ± 0.5%. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR since each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.
登录
查看更多内容
影响因子：
14.9
作者：
Guo J;Hanawalt PC;Spivak G
通讯作者：
Spivak G
影响因子：
15
作者：
Fleming AM;Orendt AM;He Y;Zhu J;Dukor RK;Burrows CJ
通讯作者：
Burrows CJ
影响因子：
64.5
作者：
Haradhvala NJ;Polak P;Stojanov P;Covington KR;Shinbrot E;Hess JM;Rheinbay E;Kim J;Maruvka YE;Braunstein LZ;Kamburov A;Hanawalt PC;Wheeler DA;Koren A;Lawrence MS;Getz G
通讯作者：
Getz G
影响因子：
56.9
作者：
Brueckner, Florian;Hennecke, Ulrich;Cramer, Patrick
通讯作者：
Cramer, Patrick
影响因子：
4.1
作者：
Durandin, Alexander;Jia, Lei;Geacintov, Nicholas E.
通讯作者：
Geacintov, Nicholas E.