van Loon Barbara, Hübscher Ulrich
Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18201-6. doi: 10.1073/pnas.0907280106. Epub 2009 Oct 9.
Reactive oxygen species (ROS) interact with DNA, frequently generating highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxo-G) lesions. Replicative DNA polymerases (pols) often misincorporate adenine opposite 8-oxo-G. The subsequent repair mechanism allowing the removal of adenine and formation of C:8-oxo-G base pair is essential to prevent C:G to A:T transversion mutations. Here, we show by immunofluorescence experiments, in cells exposed to ROS, the involvement of MutY glycosylase homologue (MUTYH) and DNA pol lambda in the repair of A:8-oxo-G mispairs. We observe specific recruitment of MUTYH, DNA pol lambda, proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN1) and DNA ligases I and III from human cell extracts to A:8-oxo-G DNA, but not to undamaged DNA. Using purified human proteins and a DNA template, we reconstitute the full pathway for the faithful repair of A:8-oxo-G mispairs involving MUTYH, DNA pol lambda, FEN1, and DNA ligase I. These results reveal a cellular response pathway to ROS, important to sustain genomic stability and modulate carcinogenesis.
活性氧（ROS）与DNA相互作用，经常产生具有高度致突变性的7,8-二氢-8-氧代鸟嘌呤（8-氧代-G）损伤。复制性DNA聚合酶（pols）常在8-氧代-G的对面错误掺入腺嘌呤。随后允许去除腺嘌呤并形成C:8-氧代-G碱基对的修复机制对于防止C:G到A:T的颠换突变至关重要。在这里，我们通过免疫荧光实验表明，在暴露于ROS的细胞中，MutY糖基化酶同源物（MUTYH）和DNA聚合酶λ参与了A:8-氧代-G错配的修复。我们观察到从人细胞提取物中，MUTYH、DNA聚合酶λ、增殖细胞核抗原（PCNA）、瓣状核酸内切酶1（FEN1）以及DNA连接酶I和III特异性募集到A:8-氧代-G DNA，但未募集到未受损的DNA。使用纯化的人蛋白和DNA模板，我们重建了涉及MUTYH、DNA聚合酶λ、FEN1和DNA连接酶I的A:8-氧代-G错配的忠实修复完整途径。这些结果揭示了一种对ROS的细胞反应途径，对维持基因组稳定性和调节致癌作用很重要。
