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Functional Conversion of CPD and (6-4) Photolyases by Mutation

机译：CPD和（6-4）光解酶通过突变的功能转化

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摘要

Ultraviolet (UV) light from the sun damages DNA by forming a cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) PP]. Photolyase (PHR) enzymes utilize near-UV/blue light for DNA repair, which is initiated by light induced electron transfer from the fully reduced flavin adenine dinucleotide chromophore. Despite similar structures and repair mechanisms, the functions of PHR are highly selective; CPD PHR repairs CPD, but not (6-4) PP, and vice versa. In this study, we attempted functional conversion between CPD and (6-4) PHRs. We found that a triple mutant of (6-4) PHR is able to repair the CPD photoproduct, though the repair efficiency is 1 order of magnitude lower than that of wild-type CPD PHR Difference Fourier transform infrared spectra for repair demonstrate the lack of secondary structural alteration in the mutant, suggesting that the triple mutant gains substrate binding ability while it does not gain the optimized conformational changes from light-induced electron transfer to the release of the repaired DNA. Interestingly, the (6-4) photoproduct is not repaired by the reverse mutation of CPD PHR, and eight additional mutations (total of 11 mutations) introduced into CPD PHR are not sufficient. The observed asymmetric functional conversion is interpreted in terms of a more complex repair mechanism for (6-4) repair, which was supported by quantum chemical/molecular mechanical calculation. These results suggest that CPD PHR may represent an evolutionary origin for photolyase family proteins.
机译：来自太阳的紫外线（UV）通过形成环丁烷嘧啶二聚体（CPD）和嘧啶（6-4）嘧啶酮光产物[（6-4）PP]破坏DNA。光解酶（PHR）酶利用近紫外/蓝光进行DNA修复，这是由光诱导的，电子从完全还原的黄素腺嘌呤二核苷酸发色团转移而引发的。尽管具有相似的结构和修复机制，但PHR的功能却具有高度选择性。 CPD PHR修复CPD，但不修复（6-4）PP，反之亦然。在这项研究中，我们尝试了CPD和（6-4）PHR之间的功能转换。我们发现（6-4）PHR的三重突变体能够修复CPD光产物，尽管其修复效率比野生型CPD PHR的修复效率低1个数量级。突变体中的二级结构改变，表明三重突变体获得了底物结合能力，而没有获得从光诱导的电子转移到修复的DNA释放的最佳构象变化。有趣的是，（6-4）光产物不能通过CPD PHR的反向突变修复，并且引入CPD PHR的另外8个突变（总共11个突变）是不够的。观察到的不对称功能转化是根据（6-4）修复的更复杂的修复机理来解释的，这由量子化学/分子力学计算来支持。这些结果表明，CPD PHR可能代表了光裂解酶家族蛋白的进化起源。

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