Abstract
Similar imperfect purine/pyrimidine mirror repeat (PMR) elements have previously been identified upstream of the human MUC1 mucin and CFTR genes. These elements confer S1 nuclease sensitivity on isolated plasmid DNA at low pH. We now present a detailed characterization of the non-B DNA structure responsible for S1 nuclease sensitivity upstream of the MUC1 gene. A approximately 90-base pair (bp) DNA fragment containing a 32-bp PMR element termed M-PMR3 was subcloned into a recombinant vector. This fragment conferred S1 nuclease sensitivity on the resulting supercoiled plasmid. High resolution mapping of sites reactive to S1 and P1 nucleases demonstrates that cleavage occurs within the M-PMR3 element. High resolution mapping with chemical agents selective for non-B DNA provides evidence that M-PMR3 adopts an H-DNA structure (intramolecular triple helix) in the less common H-y5 isomer at low pH. This result is observed in the presence or absence of Mg2+. Mutation of the native M-PMR3 element to create perfect homopurine/homopyrimidine mirror symmetry alters the preferred folding to the more common H-y3 triplex DNA isomer. These results demonstrate that imperfections in mirror symmetry can alter the relative stabilities of different H-DNA isomers.
Highlights
These elements confer S1 nuclease sensitivity on isolated plasmid DNA at low pH
S1 Nuclease Hypersensitivity in the MUC1 Promoter Maps to the M-PMR3 Element—In previous experiments, we studied the S1 nuclease sensitivity of DNA fragments derived from the CFTR and MUC1 gene promoters [9]
We sought to determine if M-PMR3 displayed S1 nuclease sensitivity when isolated from the MUC1 promoter
Summary
Subsequent high resolution studies of the PMR element upstream of the CFTR gene detected the presence of a non-B DNA structure related to the H-y3 isomer of H-DNA at low pH [13]. Single-strand-specific nuclear proteins were detected in nuclear extracts from cultured human cell lines [9]. One of these factors, a 27-kDa protein, bound preferentially to purine-rich single strands including those within the PMRs from the CFTR and MUC1 genes, suggesting the possibility that this factor could stabilize certain unpaired DNA structures. In the present study we more fully characterize the non-B DNA structure responsible for S1 nuclease sensitivity of M-PMR3
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