The i-Motif in the bcl-2 P1 Promoter Forms an Unexpectedly Stable Structure with a Unique 8:5:7 Loop Folding Pattern

Abstract

Transcriptional regulation of the bcl-2 proto-oncogene is highly complex, with the majority of transcription driven by the P1 promoter site and the interaction of multiple regulatory proteins. A guanine-and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to bcl-2 promoter activity, as deletion or mutation of this region significantly increases transcription. This GC-rich element consists of six contiguous runs of guanines and cytosines that have the potential to adopt DNA secondary structures, the G-quadruplex and i-motif, respectively. Our laboratory has previously demonstrated that the polypurine-rich strand of the bcl-2 promoter can form a mixture of three different G-quadruplex structures. In this current study, we demonstrate that the complementary polypyrimidine-rich strand is capable of forming one major intramolecular imotif DNA secondary structure with a transition pH of 6.6. Characterization of the i-motif folding pattern using mutational studies coupled with circular dichroic spectra and thermal stability analyses revealed an 8:5:7 loop conformation as the predominant structure at pH 6.1. The folding pattern was further supported by chemical footprinting with bromine. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. The potential of the GC-rich element within the bcl-2 promoter region to form DNA secondary structures suggests that the transition from the B-DNA to non-B-DNA conformation may play an important role in bcl-2 transcriptional regulation. Furthermore, the two adjacent large lateral loops in the i-motif structure provide an unexpected opportunity for protein and small molecule recognition.

Replication Fork Stalling and Checkpoint Activation by a PKD1 Locus Mirror Repeat Polypurine-Polypyrimidine (Pu-Py) Tract

“…In support of this thesis, non-B DNA structures are known to inhibit replication fork progress (18,20,64) and activate DNA damage checkpoints (35,79). Under physiological conditions, the Pu-rich strand may form G-quadruplex or triplex structures, whereas C-rich strand structures are less likely to occur in vivo as they are stabilized by hemiprotonated C-C ϩ base pairing at acid pH (62,80). Our results demonstrate that the PKD1 IVS21 Pu-Py tract can inhibit primer extension on purified DNA in vitro and in human chromosomes in a polar manner.…”

Formation and Dissociation of the Interstrand i-Motif by the Sequences d(XnC4Ym) Monitored with Electrospray Ionization Mass Spectrometry

“…However, it is now known that certain i-motifs may be stable in vivo [14,15]. There are two kinds of i-motif structures, 5′E and 3′E, determined by the end of the intercalated base pairs at the outside of the structure (Scheme1b) [12,16,17].…”

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

“…The C-rich ssDNA from S. cerevisiae meiosisspecific DSB adopts i-motif conformation Multiple studies have shown that the formation of i-motif structures by C-rich ODNs result in a characteristic retarded mobility of DNA in polyacrylamide gels, and a specific CD spectroscopic signature with a maximum positive peak between 280 and 288 nm and a negative peak within 260-267 nm (63)(64)(65)90). After having shown that the G-rich ssDNA derived from a meiosis-specific DSB folds into intramolecular GQs, we asked whether the C-rich strand complementary to the G-rich sequence can adopt i-motif conformation.…”

“…Additionally, recent studies have revealed that both GQ DNA and i-motifs are formed simultaneously within the same double-stranded tract in a variety of DNA templates (62,63). In support of their biological relevance, various studies have found that the i-motif plays a regulatory role in gene expression (64)(65)(66). Indeed, a number of proteins have been shown to bind cytosinerich strands, possibly i-motif structures.…”