Abstract
Structural variation of DNA within the promoter of the human Na, K-ATPase alpha2 gene, which contains a 35-base pair (bp) homopyrimidine.homopurine (Py.Pu) tract adjacent to a TATA box has been studied. The Py.Pu tract contains a 26-bp quasi-mirror repeat sequence with a potential for intramolecular triplex formation. As analyzed by two-dimensional agarose gel electrophoresis, a plasmid containing 151 bp of the promoter sequence including the 35-bp Py.Pu tract undergoes structural transitions under moderately acidic pH. Chemical probing with chloroacetaldehyde, dimethyl sulfate, and potassium permanganate is consistent with the formation of triplex DNA within the Py.Pu tract at native superhelical density as isolated from Escherichia coli. Chemical probing was used to determine a supercoil dependence for the formation of this combined unwound structure. At the superhelical density sufficient to locally unwind DNA, an H-y3 isomer of intermolecular triplex likely forms. However, at higher superhelical tension an H-y5 structure forms in the Py.Pu tract, and with increasing supercoiling the local DNA unwinding extends into the abutting TATA box. The H-y5/open TATA box combination structure might be favorable at higher superhelical densities since it relaxes more supercoils. The possible involvement of the H-y5/open TATA box structure in transcription is discussed.
Highlights
Homopyrimidine1⁄7homopurine (Py1⁄7Pu)1 tracts capable of forming triple-stranded DNA structures exist in the genomes of various organisms
There is an imperfect mirror repeat element within this sequence which could participate in the formation of various intramolecular triplex (H-DNA) structures
Two-dimensional gel electrophoresis is a convenient method of measuring the relaxation of superhelical tension associated with local unwinding of the DNA double helix [40]
Summary
Detailed structure-function analyses for many eukaryotic genes have demonstrated the importance of the Py1⁄7Pu sequences for promoter function. For the c-myc promoter, H-DNA formation in vitro correlated positively with the potential of the Py1⁄7Pu tract to promote the transcription in vivo [35]. The mechanisms of an influence of H-DNA forming sequences on gene expression are not yet well understood [11]. We have studied structural variation of DNA within the promoter of the human Na,K-ATPase ␣2 gene (see Fig. 1) which contains a 35-bp Py1⁄7Pu tract adjacent to a TATA box [36]. The Py1⁄7Pu tract contains a 26-bp quasi-mirror repeat sequence with a potential for H-DNA formation. The local DNA unwinding is not limited to the Py1⁄7Pu tract; the sequence of the neighboring TATA box becomes unwound as well. The H-y5 isomer is predominant, and the TATA box adjoining a single strand in this H-DNA isomer becomes unwound leading to the further relaxation of supercoils
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