Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA.
J Mol Biol. 2011 Jul 15;410(3):461-76. doi: 10.1016/j.jmb.2011.05.007. Epub 2011 May 18.
An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 Å resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 Å). These structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.
一种必需蛋白 DnaA 与复制起点 oriC 内的 9 个碱基对 DNA 位点结合。这些结合事件是形成启动复制的神秘核蛋白支架的先决条件。不同细菌 oriC 内这些短 DNA 位点或 DnaA 盒的数量、序列、位置和取向差异很大。为了研究对结核分枝杆菌 DnaA（MtDnaA）结合很重要的 DnaA 盒特征，我们已经确定了与同源 MtDnaA 盒（分辨率为 2.0 Å）和大肠杆菌共识 DnaA 盒（分辨率为 2.3 Å）结合的 MtDnaA 的 DNA 结合结构域（DBD）的晶体结构。这些结构与 MtDnaA DBD 在一系列 DnaA 盒变体中的量热平衡结合研究相结合，揭示了 DNA 识别的主要决定因素，并确立了 [T/C][T/A][G/A]TCCACA 序列为高亲和力 MtDnaA 盒。生物信息学和量热分析表明，分枝杆菌 oriC 中的 DnaA 盒序列通常与最佳结合序列不同。这种序列变化通常发生在前两个碱基对，使得体内分枝杆菌 DnaA 盒实际上是 7 个碱基对而不是 9 个碱基对。我们证明，这些 MtDnaA 盒变体与 MtDnaA DBD 的亲和力相对于最高亲和力盒 TTGTCCACA 的降低小于 10 倍。对 MtDnaA 和大肠杆菌 DnaA 的 DnaA 盒识别的理解，使得可以在结核分枝杆菌和其他真细菌的基因组中映射 DnaA 盒序列。
