Protein hnRNP A1 and its derivative Up1 unfold quadruplex DNA in the human KRAS promoter: implications for transcription

Abstract

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.

HRASis silenced by two neighboring G-quadruplexes and activated by MAZ, a zinc-finger transcription factor with DNA unfolding property

“…The affinity of MAZ-GST for the parallel hras -2 quadruplex is 2.4-fold higher than for the antiparallel hras -1 quadruplex. The K D 's between MAZ-GST and HRAS quadruplexes are not so different from those reported for the binding of recombinant UP1, hRNP A1, nucleophosmin and nucleolin to G4-DNA (26–28). Instead, the K D reported for the interaction between cellular Sp1 and quadruplex cKIT is >10-fold lower (29).…”

“…The unfolding process can be followed by fluorescence-resonance energy transfer (FRET), using hras -1 and hras -2 quadruplex-forming sequences tagged at the 5′ and 3′ ends with FAM (donor) and TAMRA (acceptor) [F- hras 1-T, F- hras 2-T] (26,30,31). In Figure 4A we report the data obtained with the antiparallel hras -1 quadruplex.…”

Probing the Potential Role of Non-B DNA Structures at Yeast Meiosis-Specific DNA Double-Strand Breaks

“…However, it is possible that specific nuclear proteins could promote and/or stabilize i-motif structures under physiological conditions in vivo (56,(67)(68)(69)(70). The formation of an i-motif structure by a C-rich motif associated with a meiosis-specific DSB at a near-neutral pH supports the notion that this structure is physiologically relevant.…”

“…Indeed, a number of proteins have been shown to bind cytosinerich strands, possibly i-motif structures. Examples include hnRNP A1 (67,68), hnRNP K (69,70), hnRNP LL (56), the Trypanosoma brucei ST-1 protein (71), and the rat nuclear protein qTBP42 (72). On the basis of the reports described above, it is apparent that i-motif structures can serve as attractive drug targets for anti-cancer therapy (56,73,74).…”

In silico screening of quadruplex-binding ligands

“…The identification of G-quadruplex-binding proteins such as nucleolin [15] and NM23-H2 [16] seems to provide strong circumstantial evidence of the existence of G-quadruplexes in living organisms. In addition to the human telomeric G-quadruplexes formed from TTAGGG repeats, bioinformatics analysis has revealed an over-representation of G-quadruplex-forming sequences in the promoter regions of genes, including oncogenes such as c-myc [17][18][19], bcl-2 [20,21], VEGF [22][23][24], KRAS [25,26], c-kit [27][28][29][30][31] and RET [32,33]. Thus, G-quadruplexes have been proposed to be intimately associated with various biological processes such as transcriptional control of gene expression and the regulation of the cell cycle and apoptosis.…”