Quadruplex–duplex competition in the nuclease hypersensitive element of human c-myc promoter: C to T mutation in C-rich strand enhances duplex association Kangkan Halder, Vidhi Mathur, Dipankar Chugh, Anjali Verma, Shantanu Chowdhury * Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110007, India Received 22 November 2004 Available online 8 December 2004 Abstract The nuclease hypersensitive element NHE III I is an important anti-cancer target as the transcription of oncogene c-myc is largely regulated by it. It has been postulated that regulatory control is mediated by G-quadruplex formation in the NHE anti-sense strand through a competition between the duplex and the quadruplex states. A mutation in the NHE has been implicated in cancer. In this study, the reported mutation has been characterized vis-a ` -vis the kinetics of i-tetraplex formation (in the sense strand) and its effect on duplex formation. We found that i-tetraplex formation was destabilized by 1.4 kcal/mol (DDG at 20 °C, pH 5.8). Observed hyster- esis allowed us to analyze the kinetics of folding for the mutant (M3). Though we observed higher association (DE on 23.4 kcal/ mol) and dissociation (DE off  22.1 kcal/mol) activation energies (at pH 5.3) for the wild-type (P1) tetraplex folding, the kinetics of folding and unfolding for M3 was somewhat faster at pH 5.3 and 5.8. Interestingly, Surface plasmon resonance (BIAcore) analysis of hybridization at pH 6.6 indicated a higher association constant for M3 (22.5 · 10 4 M 1 s 1 ) than P1 (3.2 · 10 4 M 1 s 1 ). The equi- librium dissociation constants also indicated favorable duplex association for M3 (22.2 and 190.6 nM for M3 and P1, respec- tively). We envisage that the increased affinity for the duplex state due to the mutation could play a functional role in the aberrant regulation of c-myc. Ó 2004 Elsevier Inc. All rights reserved. Keywords: i-Motif; NM23-H2; Hysteresis; Surface plasmon resonance; BIAcore; Hybridization kinetics Human c-myc has been implicated in many malignant tumors [1,2]. c-myc expression is positively regulated by a promoter element designated nuclease hypersensitive ele- ment (NHE III 1 ) which corresponds to bases 2180–2212 in the human c-myc locus [3,4]. The NHE III 1 is guanine- rich in its anti-sense strand while the complementary sense strand is cytosine-rich. A secondary structure formed by the G-rich sequence in the NHE—the G-quadruplex has been implicated as a factor in regula- tion of c-myc [5,6]. Thus, postulated models of regulation entail a switching between the structural motif and du- plex DNA [5,6]. In this context, the role of the comple- mentary C-rich sense strand is less clearly understood; despite the observation that this strand adopts a stable tetraplex structure (i-motif) in solution [7]. C-tetraplexes are formed by the base pairing of hemiprotonated cyto- sine + and cytosine to form duplex structures, which are intercalated in an antiparallel orientation to form tetra- plexes [8]. A pH-dependent equilibrium involving mono- meric i-tetraplexes was first observed in a 17-mer C-rich fragment from human centromeric satellite III [9]. Intra- molecular i-motifs were also observed in multiple dode- camer repeats implicated in progressive myoclonus epilepsy [10]. We recently found that the thermodynamic properties of i-tetraplex folding from c-myc NHE are 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.137 * Corresponding author. Fax: +91 11 2766 7471. E-mail address: shantanuc@igib.res.in (S. Chowdhury). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 327 (2005) 49–56 BBRC