Potential for H-DNA in the Human Mucin Gene Promoter

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 ϳ90-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 Mg 2؉. 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.

Downregulation of Muc1 in MMTV-c-Neu tumors

“…Further, this hypersensitive site was also present in transgenic mice expressing the human gene, suggesting that mice express the trans-acting factors that potentially bind to this site (Shiraga et al, 2002). In the human gene, this hypersensitive site contains a pyrimidine mirror repeat (PMR) that is associated with the formation of H-DNA (Nelson et al, 1996). Regions of DNA that contain polypyrimidine or polypurine tracts involving a mirror repeat have the capability of forming a triplex structure known as H-DNA (Mirkin et al, 1987).…”

Naturally occurring H-DNA-forming sequences are mutagenic in mammalian cells

“…All H-DNA-forming sequences used have been shown to adopt H-DNA conformations with different efficiencies in previous studies (12,23). To ensure that these sequences were able to adopt H-DNA structures in the newly constructed plasmids, we examined the formation of H-DNA structures by using S1 and P1 nuclease sensitivity assays at pH 4.5 and 7.1, respectively, as described (24), with slight modification. After nuclease digestion, the 5Ј ends produced at the single-stranded region in the H-DNA loci were radiolabeled with T4 polynucleotide kinase and [␥-32 P]ATP.…”

Activation of STAT3 by the c-Fes Protein-tyrosine Kinase

“…The mixture was incubated at room temperature for 30 min. and the unincorporated nucleotides were removed by spermine precipitation (28). DNA binding reactions contained 40,000 cpm of labeled probe in a final volume of 20 l and 2-6 g of cytosolic protein extract.…”