High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation

Abdelaziz Kerkour,Julien Marquevielle,Stefaniia Ivashchenko,Liliya A Yatsunyk,Jean-Louis Mergny,Gilmar F Salgado

doi:10.1074/jbc.m117.781906

Abstract

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression.

Highlights

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4)
Certain G4 structures in this region are stabilized by G-quadruplex-interacting ligands [12, 13, 43, 49] such as guanidine-modified phthalocyanines that interfere with KRAS transcription by competing with Myc-associated zinc finger (MAZ) and poly(ADP-ribose) polymerase 1 proteins
The G4 conformation revealed by our studies provides a model that could potentially be used for in silico drug screening for ligands that stabilize the G4 structure in the KRAS promoter

Summary

Introduction

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5؅-AGGGCGGTGTGGGAATAGGGAA-3؅) and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. 32R contains six guanine stretches and is able to form several G4 conformations Among those stretches of guanines, two regions overlap and includes the Myc-associated zinc finger (MAZ)-binding sites, the recognition sequence for a transcription factor that recognizes GGGCGG and GGGAGG sequences [46]. The approach targeting unusual motifs present in genomic DNA is actively being pursued and can be seen as a new alternative strategy with promising results [1, 28, 29, 41]

Methods

Results

Conclusion