Cogoi Susanna, Xodo Luigi E
Department of Biomedical Science and Technology, School of Medicine, P.le Kolbe 4, 33100 Udine, Italy.
Nucleic Acids Res. 2006 May 10;34(9):2536-49. doi: 10.1093/nar/gkl286. Print 2006.
In human and mouse, the promoter of the KRAS gene contains a nuclease hypersensitive polypurine-polypyrimidine element (NHPPE) that is essential for transcription. An interesting feature of the polypurine G-rich strand of NHPPE is its ability to assume an unusual DNA structure that, according to circular dichroism (CD) and DMS footprinting experiments, is attributed to an intramolecular parallel G-quadruplex, consisting of three G-tetrads and three loops. The human and mouse KRAS NHPPE G-rich strands display melting temperature of 64 and 73 degrees C, respectively, as well as a K+-dependent capacity to arrest DNA polymerase. Photocleavage and CD experiments showed that the cationic porphyrin TMPyP4 stacks to the external G-tetrads of the KRAS quadruplexes, increasing the T(m) by approximately 20 degrees C. These findings raise the intriguing question that the G-quadruplex formed within the NHPPE of KRAS may be involved in the regulation of transcription. Indeed, transfection experiments showed that the activity of the mouse KRAS promoter is reduced to 20% of control, in the presence of the quadruplex-stabilizing TMPyP4. In addition, we found that G-rich oligonucleotides mimicking the KRAS quadruplex, but not the corresponding 4-base mutant sequences or oligonucleotides forming quadruplexes with different structures, competed with the NHPPE duplex for binding to nuclear proteins. When vector pKRS-413, containing CAT driven by the mouse KRAS promoter, and KRAS quadruplex oligonucleotides were co-transfected in 293 cells, the expression of CAT was found to be downregulated to 40% of the control. On the basis of these data, we propose that the NHPPE of KRAS exists in equilibrium between a double-stranded form favouring transcription and a folded quadruplex form, which instead inhibits transcription. Such a mechanism, which is probably adopted by other growth-related genes, provides useful hints for the rational design of anticancer drugs against the KRAS oncogene.
在人类和小鼠中，KRAS基因的启动子包含一个核酸酶超敏多嘌呤-多嘧啶元件（NHPPE），它对转录至关重要。NHPPE富含多嘌呤的G链的一个有趣特征是其能够呈现一种不寻常的DNA结构，根据圆二色性（CD）和二甲基亚砜足迹实验，这归因于一种分子内平行G-四链体，由三个G-四联体和三个环组成。人类和小鼠的KRAS NHPPE富含G的链分别显示出64和73摄氏度的解链温度，以及依赖K + 阻止DNA聚合酶的能力。光裂解和CD实验表明，阳离子卟啉TMPyP4堆积在KRAS四链体的外部G-四联体上，使解链温度（T m）提高了约20摄氏度。这些发现提出了一个有趣的问题，即KRAS的NHPPE内形成的G-四链体可能参与转录调控。事实上，转染实验表明，在存在四链体稳定剂TMPyP4的情况下，小鼠KRAS启动子的活性降低至对照的20%。此外，我们发现模拟KRAS四链体的富含G的寡核苷酸，而不是相应的4碱基突变序列或形成不同结构四链体的寡核苷酸，与NHPPE双链体竞争结合核蛋白。当包含由小鼠KRAS启动子驱动的CAT的载体pKRS-413和KRAS四链体寡核苷酸在293细胞中共转染时，发现CAT的表达下调至对照的40%。基于这些数据，我们提出KRAS的NHPPE以有利于转录的双链形式和抑制转录的折叠四链体形式之间的平衡存在。这种机制可能为其他与生长相关的基因所采用，为针对KRAS癌基因的抗癌药物的合理设计提供了有用的线索。
