Chen Si-Han, Suzuki Carolyn K, Wu Shih-Hsiung
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
Nucleic Acids Res. 2008 Mar;36(4):1273-87. doi: 10.1093/nar/gkm1140. Epub 2008 Jan 3.
Lon is an ATP-powered protease that binds DNA. However, the function of DNA binding by Lon remains elusive. Studies suggest that human Lon (hLon) binds preferentially to a G-rich single-stranded DNA (ssDNA) sequence overlapping the light strand promoter of mitochondrial DNA. This sequence is contained within a 24-base oligonucleotide referred to as LSPas. Here, we use biochemical and biophysical approaches to elucidate the structural properties of ssDNAs bound by hLon, as well as the thermodynamics of DNA binding by hLon. Electrophoretic mobility shift assay and circular dichroism show that ssDNAs with a propensity for forming parallel G-quartets are specifically bound by hLon. Isothermal titration calorimetry demonstrates that hLon binding to LSPas is primarily driven by enthalpy change associated with a significant reduction in heat capacity. Differential scanning calorimetry pinpoints an excess heat capacity upon hLon binding to LSPas. By contrast, hLon binding to an 8-base G-rich core sequence is entropically driven with a relatively negligible change in heat capacity. A considerable enhancement of thermal stability accompanies hLon binding to LSPas as compared to the G-rich core. Taken together, these data support the notion that hLon binds G-quartets through rigid-body binding and that binding to LSPas is coupled with structural adaptation.
Lon是一种由ATP供能的蛋白酶，可结合DNA。然而，Lon与DNA结合的功能仍不清楚。研究表明，人类Lon（hLon）优先结合与线粒体DNA轻链启动子重叠的富含G的单链DNA（ssDNA）序列。该序列包含在一个被称为LSPas的24个碱基的寡核苷酸中。在这里，我们使用生化和生物物理方法来阐明hLon结合的ssDNA的结构特性，以及hLon与DNA结合的热力学。电泳迁移率变动分析和圆二色性表明，倾向于形成平行G-四联体的ssDNA被hLon特异性结合。等温滴定量热法表明，hLon与LSPas的结合主要由与热容显著降低相关的焓变驱动。差示扫描量热法指出hLon与LSPas结合时存在过量的热容。相比之下，hLon与一个8碱基富含G的核心序列的结合是由熵驱动的，热容变化相对可忽略不计。与富含G的核心序列相比，hLon与LSPas的结合伴随着热稳定性的显著提高。综上所述，这些数据支持了hLon通过刚体结合与G-四联体结合且与LSPas的结合伴随着结构适应的观点。
