Protein hnRNP A1 and its derivative Up1 unfold quadruplex DNA in the human KRAS promoter: implications for transcription.
The promoter of the human KRAS proto-oncogene contains a structurally polymorphic nuclease hypersensitive element (NHE) whose purine strand forms a parallel G-quadruplex structure (called 32R). In a previous work we reported that quadruplex 32R is recognized by three nuclear proteins: PARP-1, Ku70 and hnRNP A1. In this study we describe the interaction of recombinant hnRNP A1 (A1) and its derivative Up1 with the KRAS G-quadruplex. Mobility-shift experiments show that A1/Up1 binds specifically, and also with a high affinity, to quadruplex 32R, while CD demonstrates that the proteins strongly reduce the intensity of the 260 nm-ellipticity-the hallmark for parallel G4-DNA-and unfold the G-quadruplex. Fluorescence resonance energy transfer melting experiments reveal that A1/Up1 completely abrogates the cooperative quadruplex-to-ssDNA transition that characterizes the KRAS quadruplex and facilitates the association between quadruplex 32R and its complementary polypyrimidine strand. When quadruplex 32R is stabilized by TMPyP4, A1/Up1 brings about only a partial destabilization of the G4-DNA structure. The possible role played by hnRNP A1 in the mechanism of KRAS transcription is discussed.
Paramasivam
Manikandan
M
Department of Biomedical Science and Technology, School of Medicine, Udine, Italy.
Membrino
Alexandro
A
Cogoi
Susanna
S
Fukuda
Hirokazu
H
Nakagama
Hitoshi
H
Xodo
Luigi E
LE
D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D031541:Fluorescence Resonance Energy Transfer; D054856:G-Quadruplexes; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism*; D006801:Humans; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D014158:Transcription, Genetic; D018631:ras Proteins / Q000235:genetics*
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C117307:KRAS protein, human; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; C000619656:hnRNPA1 protein, human; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins