Abstract
Enrichment of four tandem repeats of guanine (G) rich and cytosine (C) rich sequences in functionally important regions of human genome forebodes the biological implications of four-stranded DNA structures, such as G-quadruplex and i-motif, that can form in these sequences. However, there have been few reports on the intramolecular formation of non-B DNA structures in less than four tandem repeats of G or C rich sequences. Here, using mechanical unfolding at the single-molecule level, electrophoretic mobility shift assay (EMSA), circular dichroism (CD), and ultraviolet (UV) spectroscopy, we report an intramolecularly folded non-B DNA structure in three tandem cytosine rich repeats, 5'-TGTC4ACAC4TGTC4ACA (ILPR-I3), in the human insulin linked polymorphic region (ILPR). The thermal denaturation analyses of the sequences with systematic C to T mutations have suggested that the structure is linchpinned by a stack of hemiprotonated cytosine pairs between two terminal C4 tracts. Mechanical unfolding and Br2 footprinting experiments on a mixture of the ILPR-I3 and a 5′-C4TGT fragment have further indicated that the structure serves as a building block for intermolecular i-motif formation. The existence of such a conformation under acidic or neutral pH complies with the strand-by-strand folding pathway of ILPR i-motif structures.
Highlights
IntroductionNon-B DNA structures, such as Gquadruplex, i-motif, DNA cruciform, and H-DNA, can form in these tandem repeats [3]
Tandem repeats of DNA residues are abundant throughout human genome [1,2,3]
circular dichroism (CD) spectroscopy demonstrated that structures in the insulin linked polymorphic region (ILPR)-I3 sequence contain hemiprotonated cytosine pair stackings First, we performed CD measurements to determine whether stable structures exist in the wild-type ILPR sequence, 5’TGTC4ACAC4TGTC4ACA-3’ (ILPR-I3, Table 1)
Summary
Non-B DNA structures, such as Gquadruplex, i-motif, DNA cruciform, and H-DNA, can form in these tandem repeats [3] Under physiological conditions, these non-B DNA structures can prevail over DNA duplex to regulate DNA processing or gene expression. Four tandem repeats of cytosine and guanine rich sequences can host i-motif and Gquadruplex structures, respectively [6,7] The fact that these structures can form in promoter regions has led to the hypotheses that G-quadruplexes or i-motifs may regulate RNA transcriptions [8]. In addition to Human genome, C-rich repeats are found in Drosophila virilis DNA [21] and some cardiovirus RNAs [22] These discoveries expose the i-motif to a broad range of host species for potential biological roles
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