Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins.
The human KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) upstream of the major transcription initiation site. In this article, we demonstrate by primer-extension experiments, PAGE, chemical footprinting, CD, UV and FRET experiments that the G-rich strand of NHE (32R) folds into intra-molecular G-quadruplex structures. Fluorescence data show that 32R in 100 mM KCl melts with a biphasic profile, showing the formation of two distinct G-quadruplexes with T(m) of approximately 55 degrees C (Q(1)) and approximately 72 degrees C (Q(2)). DMS-footprinting and CD suggest that Q(1) can be a parallel and Q(2) a mixed parallel/antiparallel G-quadruplex. When dsNHE (32R hybridized to its complementary) is incubated with a nuclear extract from Panc-1 cells, three DNA-protein complexes are observed by EMSA. The complex of slower mobility is competed by quadruplex 32R, but not by mutant oligonucleotides, which cannot form a quadruplex structure. Using paramagnetic beads coupled with 32R, we pulled down from the Panc-1 extract proteins with affinity for quadruplex 32R. One of these is the heterogeneous nuclear ribonucleoprotein A1, which was previously reported to unfold quadruplex DNA. Our study suggests a role of quadruplex DNA in KRAS transcription and provides the basis for the rationale design of molecular strategies to inhibit the expression of KRAS.
Cogoi
Susanna
S
Department of Biomedical Science and Technology, School of Medicine, Ple. Kolbe 4, 33100 Udine and CRIBI Biotechnology Centre, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.
Paramasivam
Manikandan
M
Spolaore
Barbara
B
Xodo
Luigi E
LE
D001665:Binding Sites; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003851:Deoxyribonucleases; D054856:G-Quadruplexes; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D009691:Nucleic Acid Denaturation; D011166:Porphyrins / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic / Q000187:drug effects; D018631:ras Proteins / Q000235:genetics*
D004268:DNA-Binding Proteins; C117307:KRAS protein, human; C000715470:MAS1 protein, human; D009687:Nuclear Proteins; D011166:Porphyrins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D003851:Deoxyribonucleases; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins