Dimitri Alexandra, Goodenough Angela K, Guengerich F Peter, Broyde Suse, Scicchitano David A
Department of Biology, New York University, New York, NY 10003, USA.
J Mol Biol. 2008 Jan 11;375(2):353-66. doi: 10.1016/j.jmb.2007.10.057. Epub 2007 Oct 30.
The DNA lesion 1,N(2)-ethenoguanine (1,N(2)-epsilon G) is formed endogenously as a by-product of lipid peroxidation or by reaction with epoxides that result from the metabolism of the industrial pollutant vinyl chloride, a known human carcinogen. DNA replication past 1,N(2)-epsilon G and site-specific mutagenesis studies on mammalian cells have established the highly mutagenic and genotoxic properties of the damaged base. However, there is as yet no information on the processing of this lesion during transcription. Here, we report the results of transcription past a site-specifically modified 1,N(2)-epsilon G DNA template. This lesion contains an exocyclic ring obstructing the Watson-Crick hydrogen-bonding edge of guanine. Our results show that 1,N(2)-epsilon G acts as a partial block to the bacteriophage T7 RNA polymerase (RNAP), which allows nucleotide incorporation in the growing RNA with the selectivity A>G>(C=-1 deletion)>>U. In contrast, 1,N(2)-epsilon G poses an absolute block to human RNAP II elongation, and nucleotide incorporation opposite the lesion is not observed. Computer modeling studies show that the more open active site of T7 RNAP allows lesion bypass when the 1,N(2)-epsilon G adopts the syn-conformation. This orientation places the exocyclic ring in a collision-free empty pocket of the polymerase, and the observed base incorporation preferences are in agreement with hydrogen-bonding possibilities between the incoming nucleotides and the Hoogsteen edge of the lesion. On the other hand, in the more crowded active site of the human RNAP II, the modeling studies show that both syn- and anti-conformations of the 1,N(2)-epsilon G are sterically impermissible. Polymerase stalling is currently believed to trigger the transcription-coupled nucleotide excision repair machinery. Thus, our data suggest that this repair pathway is likely engaged in the clearance of the 1,N(2)-epsilon G from actively transcribed DNA.
DNA损伤1,N(2)-乙烯基鸟嘌呤(1,N(2)-εG)是脂质过氧化的内源性副产物，或与工业污染物氯乙烯代谢产生的环氧化物反应形成的，氯乙烯是一种已知的人类致癌物。对哺乳动物细胞进行的DNA复制越过1,N(2)-εG以及位点特异性诱变研究，证实了该损伤碱基具有高度诱变和基因毒性。然而，关于转录过程中该损伤的处理尚无相关信息。在此，我们报告了转录越过位点特异性修饰的1,N(2)-εG DNA模板的结果。该损伤含有一个外环，阻碍了鸟嘌呤的沃森-克里克氢键边缘。我们的结果表明，1,N(2)-εG对噬菌体T7 RNA聚合酶(RNAP)起到部分阻滞作用，使得核苷酸以A>G>(C=-1缺失)>>U的选择性掺入正在生长的RNA中。相比之下，1,N(2)-εG对人类RNAP II的延伸构成绝对阻滞，未观察到损伤位点对面的核苷酸掺入。计算机模拟研究表明，当1,N(2)-εG采取顺式构象时，T7 RNAP更开放的活性位点允许损伤绕过。这种取向将外环置于聚合酶无碰撞的空口袋中，观察到的碱基掺入偏好与进入的核苷酸和损伤的Hoogsteen边缘之间的氢键可能性一致。另一方面，在人类RNAP II更拥挤的活性位点，模拟研究表明1,N(2)-εG 的顺式和反式构象在空间上都是不允许的。目前认为聚合酶停滞会触发转录偶联核苷酸切除修复机制。因此，我们的数据表明，该修复途径可能参与了从活跃转录DNA中清除1,N(2)-εG的过程。
