The S1-sensitive Form of D(C-T) _n ⋅d(A-G) _n : Chemical Evidence for a Three-Stranded Structure in Plasmids

Abstract

Homopurine-homopyrimidine sequences that flank certain actively transcribed genes are hypersensitive to single strand-specific nucleases such as S1. This has raised the possibility that an unusual structure exists in these regions that might be involved in recognition or regulation. Several of these sequences, including d(C-T)n.d(A-G)n, are known to undergo a transition in plasmids to an underwound state that is hypersensitive to single strand-specific nucleases; this transition occurs under conditions of moderately acid pH and negative supercoiling. Chemical probes were used to examine the reactivity of a restriction fragment from a human U1 gene containing the sequence d(C-T)18.d(A-G)18 as a function of supercoiling and pH, and thus analyze the structure in this region. Hyperreactivity was seen in the center and at one end of the (C-T)n tract, and continuously from the center to the same end of the (A-G)n tract, in the presence of supercoiling and pH less than or equal to 6.0. These results provide strong support for a triple-helical model recently proposed for these sequences and are inconsistent with other proposed structures.

Characterization of a polypurine/polypyrimidine sequence upstream of the mouse metallothionein-I gene

“…d(GA) [16][17][18] , (14,17,20,36); d(G) 30 , (37) and other di-, tri-or tetra-nucleotide repeats, (22,38)]. In this work we wished to characterize aspects of the structure and any detectable function of a remarkable homopurine/homopyrimidine sequence located upstream of the MT-I gene.…”

“…Chemical probes that react with unpaired or unstacked bases have previously been shown to be of value in detecting and evaluating non-B DNA structures (18)(19)(20)(21)(22)(23). CAA reacts with adenine and cytosine to form their respective etheno derivatives when these bases occur in unpaired or unstacked conformations.…”

“…These regions of base unpairing are sensitive to chemical and enzymatic probes that react differentially with single-stranded DNA. Chemical and enzymatic probing allows structural information to be deduced for such sequence elements (18)(19)(20)(21)(22)(23). Stable H-DNA structures require some degree of mirror symmetry and are typically stabilized by negative supercoiling.…”

“…Certain PMRs have been shown to isomerize to H-DNA in vitro under conditions of negative supercoiling and acidic pH (11,(14)(15)(16)(17). H-DNA refers to a family of DNA structures characterized by an intramolecular DNA triplex and accompanying regions of base unpairing.…”

In Vitro and in Vivo Ligation-mediated Polymerase Chain Reaction Analysis of a Polypurine/Polypyrimidine Sequence Upstream of the Mousemetallothionein-I Gene

“…The best studied examples of R/Y DNA sequences capable of adopting H-DNA structures in vitro possess perfect mirror symmetry and are often repetitive in nature (8,9,(45)(46)(47)(48)(49)(50). In contrast, the MT-I R/Y sequence is a 128-bp R/Y sequence composed of multiple imperfect mirror repeats, but lacking any substantial region of perfect mirror symmetry.…”