Nucleic Acids Research Advance Access published online on May 19, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn120
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Molecular Biology |
Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins
1Department of Biomedical Science and Technology, School of Medicine, Ple. Kolbe 4, 33100 Udine and 2CRIBI Biotechnology Centre, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy
* To whom correspondence should be addressed. Tel: +39 0432 494395; Fax: +39 0432 494301; Email: lxodo{at}makek.dstb.uniud.it
Received January 21, 2008. Revised March 3, 2008. Accepted March 4, 2008.
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 Tm of 
55°C (Q1) and 72°C (Q2). DMS-footprinting and CD suggest that Q1 can be a parallel and Q2 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.