Baer M, Sancar G B
Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7260.
Mol Cell Biol. 1989 Nov;9(11):4777-88. doi: 10.1128/mcb.9.11.4777-4788.1989.
DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. The results of nucleotide sequence analysis and spectroscopic studies demonstrated that photolyases from Saccharomyces cerevisiae and Escherichia coli share 37% amino acid sequence homology and contain identical chromophores. Do the similarities between these two enzymes extend to their interactions with DNA containing pyrimidine dimers, or does the organization of DNA into nucleosomes in S. cerevisiae necessitate alternative or additional recognition determinants? To answer this question, we used chemical and enzymatic techniques to identify the contacts made on DNA by S. cerevisiae photolyase when it is bound to a pyrimidine dimer and compared these contacts with those made by E. coli photolyase and by a truncated derivative of the yeast enzyme when bound to the same substrate. We found evidence for a common set of interactions between the photolyases and specific phosphates in the backbones of both strands as well as for interactions with bases in both the major and minor grooves of dimer-containing DNA. Superimposed on this common pattern were significant differences in the contributions of specific contacts to the overall binding energy, in the interactions of the enzymes with groups on the complementary strand, and in the extent to which other DNA-binding proteins were excluded from the region around the dimer. These results provide strong evidence both for a conserved dimer-binding motif and for the evolution of new interactions that permit photolyases to also act as accessory proteins in nucleotide excision repair. The locations of the specific contacts made by the yeast enzyme indicate that the mechanism of nucleotide excision repair in this organism involves incision(s) at a distance from the pyrimidine dimer.
DNA光解酶催化DNA中嘧啶二聚体的光依赖性修复。核苷酸序列分析和光谱研究结果表明，酿酒酵母和大肠杆菌的光解酶具有37%的氨基酸序列同源性，并含有相同的发色团。这两种酶之间的相似性是否延伸到它们与含有嘧啶二聚体的DNA的相互作用，或者酿酒酵母中DNA组装成核小体是否需要替代或额外的识别决定因素？为了回答这个问题，我们使用化学和酶学技术来确定酿酒酵母光解酶与嘧啶二聚体结合时在DNA上形成的接触，并将这些接触与大肠杆菌光解酶以及酵母酶的截短衍生物与相同底物结合时形成的接触进行比较。我们发现了光解酶与两条链主链中特定磷酸基团之间存在一组共同相互作用的证据，以及与含有二聚体的DNA的大沟和小沟中的碱基相互作用的证据。在这种共同模式之上，特定接触对整体结合能的贡献、酶与互补链上基团的相互作用以及其他DNA结合蛋白被排除在二聚体周围区域的程度存在显著差异。这些结果为保守的二聚体结合基序以及允许光解酶在核苷酸切除修复中也作为辅助蛋白的新相互作用的进化提供了有力证据。酵母酶形成的特定接触的位置表明，该生物体中核苷酸切除修复的机制涉及在距嘧啶二聚体一定距离处进行切割。
