Lin Y, Dötsch V, Wintner T, Peariso K, Myers L C, Penner-Hahn J E, Verdine G L, Wagner G
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Biochemistry. 2001 Apr 10;40(14):4261-71. doi: 10.1021/bi002109p.
The Escherichia coli protein Ada specifically repairs the S(p) diastereomer of DNA methyl phosphotriesters in DNA by direct and irreversible transfer of the methyl group to its own Cys 69 which is part of a zinc-thiolate center. The methyl transfer converts Ada into a transcriptional activator that binds sequence-specifically to promoter regions of its own gene and other methylation resistance genes. Ada thus acts as a chemosensor to activate repair mechanisms in situations of methylation damage. Here we present a highly refined solution structure of the 10 kDa N-terminal domain, N-Ada10, which reveals structural details of the nonspecific DNA interaction of N-Ada10 during the repair process and provides a basis for understanding the mechanism of the conformational switch triggered by methyl transfer. To further elucidate this, EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near-edge structure) data were acquired, which confirmed that the zinc-thiolate center is maintained when N-Ada is methylated. Thus, ligand exchange is not the mechanism that enhances sequence-specific DNA binding and transcriptional activation upon methylation of N-Ada. The mechanism of the switch was further elucidated by recording NOESY spectra of specifically labeled methylated-Ada/DNA complexes, which showed that the transferred methyl group makes many contacts within N-Ada but none with the DNA. This implies that methylation of N-Ada induces a structural change, which enhances the promoter affinity of a remodeled surface region that does not include the transferred methyl group.
大肠杆菌蛋白Ada通过将甲基直接且不可逆地转移至自身的半胱氨酸69（它是锌硫醇盐中心的一部分），特异性修复DNA中的DNA甲基磷酸三酯的S(p)非对映异构体。甲基转移将Ada转变为一种转录激活因子，它能序列特异性地结合自身基因及其他抗甲基化基因的启动子区域。因此，Ada在甲基化损伤情况下作为一种化学传感器来激活修复机制。在此，我们展示了10 kDa N端结构域N-Ada10的高度精细的溶液结构，它揭示了修复过程中N-Ada10与非特异性DNA相互作用的结构细节，并为理解甲基转移引发的构象转换机制提供了基础。为进一步阐明这一点，获取了扩展X射线吸收精细结构（EXAFS）和X射线吸收近边结构（XANES）数据，这些数据证实当N-Ada甲基化时锌硫醇盐中心得以维持。因此，配体交换并非N-Ada甲基化后增强序列特异性DNA结合和转录激活的机制。通过记录特异性标记的甲基化-Ada/DNA复合物的核Overhauser效应光谱（NOESY）进一步阐明了转换机制，该光谱表明转移的甲基在N-Ada内部形成许多接触，但与DNA没有接触。这意味着N-Ada的甲基化诱导了一种结构变化，增强了一个不包括转移甲基的重塑表面区域对启动子的亲和力。
