Department of Physics, Programs of Biophysics, Chemical Physics, and Biochemistry, Ohio State University, 191 West Woodruff Avenue, Columbus, OH 43210, USA.
Proc Natl Acad Sci U S A. 2011 Sep 6;108(36):14831-6. doi: 10.1073/pnas.1110927108. Epub 2011 Jul 29.
Photolyase uses blue light to restore the major ultraviolet (UV)-induced DNA damage, the cyclobutane pyrimidine dimer (CPD), to two normal bases by splitting the cyclobutane ring. Our earlier studies showed that the overall repair is completed in 700 ps through a cyclic electron-transfer radical mechanism. However, the two fundamental processes, electron-tunneling pathways and cyclobutane ring splitting, were not resolved. Here, we use ultrafast UV absorption spectroscopy to show that the CPD splits in two sequential steps within 90 ps and the electron tunnels between the cofactor and substrate through a remarkable route with an intervening adenine. Site-directed mutagenesis reveals that the active-site residues are critical to achieving high repair efficiency, a unique electrostatic environment to optimize the redox potentials and local flexibility, and thus balance all catalytic reactions to maximize enzyme activity. These key findings reveal the complete spatio-temporal molecular picture of CPD repair by photolyase and elucidate the underlying molecular mechanism of the enzyme's high repair efficiency.
光解酶利用蓝光将主要的紫外线（UV）诱导的 DNA 损伤——环丁烷嘧啶二聚体（CPD）——通过分裂环丁烷环恢复为两个正常碱基。我们之前的研究表明，通过循环电子转移自由基机制，整体修复在 700 皮秒内完成。然而，两个基本过程，电子隧穿途径和环丁烷环的分裂，尚未得到解决。在这里，我们使用超快紫外吸收光谱表明，CPD 在 90 皮秒内分两步分裂，电子通过一个具有 intervening adenine 的显著途径在辅因子和底物之间隧穿。定点突变揭示了活性位点残基对于实现高效率修复至关重要，独特的静电环境可优化氧化还原电势和局部灵活性，从而平衡所有催化反应以最大限度地提高酶活性。这些关键发现揭示了光解酶修复 CPD 的完整时空分子图像，并阐明了该酶高效修复的潜在分子机制。
