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Nucleic Acids Research Advance Access originally published online on May 19, 2008
Nucleic Acids Research 2008 36(11):3765-3780; doi:10.1093/nar/gkn120
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Nucleic Acids Research, 2008, Vol. 36, No. 11 3765-3780
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins

Susanna Cogoi1, Manikandan Paramasivam1, Barbara Spolaore2 and Luigi E. Xodo1,*

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.


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