Magnesium ion-dependent triple-helix structure formed by homopurine-homopyrimidine sequences in supercoiled plasmid DNA.

Abstract

DNA can be chemically cleaved at the site of chloroacetaldehyde-modified residues by the chemicals used for Maxam-Gilbert sequencing reactions. Use of this technique facilitates fine structural analysis of unpaired DNA bases in DNA with non-B-DNA structure. This method was used to study the non-B-DNA structure adopted by the poly-(dG)-poly(dC) sequence under torsional stress at various ionic conditions. In the presence of 2 mM Mg2+, the 5' half of the deoxycytosine tract is very reactive to chloroacetaldehyde, while the 3' half is virtually unreactive. In the poly(dG) tract, chloroacetaldehyde reaction is restricted to the center guanine residues. In the absence of Mg2+, however, it is the 5' halfofthe deoxyguanine tract that is reactive to chloroacetaldehyde, while the 3' half is unreactive. And chloroacetaldehyde reaction is restricted to the center cytosine residues in the poly(dC) stretch. These results strongly suggest that the poly(dG)-poly(dC) sequence is folded into halves from the center of the sequence to form a tetra-stranded-like structure. Such a structure contains either a triplex consisting of poly(dG) poly(dG) poly(dC) strands in the presence of Mg2+ or a triplex consisting of poly(dC)-poly(dG)'poly(dC) strands in the absence of Mg2+. The fourth strand, not involved in triplex formation, is closely associated with the triplex and is positioned in such a way that DNA bases are exposed and freely accessible to the chloroacetaldehyde reaction.The torsional stress that results from negative supercoiling is relieved through the formation of non-B-form DNA structures. Under torsional stress, non-B-DNA structures are preferentially formed by certain DNA sequences. Homopurine/homopyrimidine sequences are one type of such DNA sequences known to adopt non-B-DNA structure in supercoiled DNA. Long stretches of homopurine/homopyrimidine are often found in putative regulatory regions of eukaryotic genes. The anomalous sensitivity of these homopurine/homopyrimidine sequences to S1 nuclease, when studied in supercoiled DNA, has been reported by a number of groups (1-9).We previously devised a method that used bromoacetaldehyde and chloroacetaldehyde to detect and analyze the altered DNA conformation of chromatin in vivo and in supercoiled DNA (10-12). Both bromoacetaldehyde and its less potent analog chloroacetaldehyde react specifically at the N-1 and N-6 positions of adenine and the N-3 and N4 positions of cytosine residues (13-16), when these bases are not hydrogen bonded (17, 18). Although less reactive, the N-1 and N-2 positions of guanine residues also react with chloroacetaldehyde (19). Using bromoacetaldehyde as the probe and then digesting the bromoacetaldehyde-modified sites with S1 nuclease after cleaving the DNA with a restriction enzyme, we have shown that the 16 contiguous guanine residues at -180 base pairs (bp) from the mRNA cap site of the chicken adult 3A-globin gene constitute one of the major sites detected by bromoacetaldehyde in supercoiled DNA but not in linear DNA (10). Furthermor...

Characterization of the DNA triplex formed by d(TGGGTGGGTGGTTGGGTGGG) and a critical R · Y sequence located in the promoter of the murine Ki‐ras proto‐oncogene

“…Thereafter, Kohwi and KowhiShigematzu demonstrated that a segment of poly dG. poly(dC), if inserted in a supercoiled plasmid, adopted in the presence of Mf + an intramolecular structure stabilised by G'GC triads [23].…”

Molecular characterization of a common fragile site ( FRA7H ) on human chromosome 7 by the cloning of a simian virus 40 integration site

“…Non-B-DNA structures were detected by chloroacetaldehyde (CAA) in vitro (29). In brief, supercoiled plasmid DNA was reacted with 2 l of doubledistilled CAA in a 100-l reaction volume at 37°C with or without 2 mM MgCl 2 at pH 7 (50 mM Tris⅐HCl).…”

“…CAA has been used for non-B-DNA structure analysis (48). CAA reaction is ideal for detecting only unpaired bases (even only a single unpaired base) within double-stranded DNA (29). In vitro analysis of triple-helix formation was performed on a 4.4-kb EcoRI fragment, cloned from cosmid 137g12 (base position 106,853-111,288) (Fig.…”

Conformational junctions between left‐handed DNA in (dA‐dT)16 and contiguous B‐DNA in a supercoiled plasmid contain chemically reactive bases

“…This difference was not due to reduced CAA induced reactivity of bases to formic acid: native DNA modified with CAA in 2.0 M NaCl undergoes pronounced modification only in cruciform loop regions in DNA reacted with either hydrazine or formic acid (not shown), though the latter is less 3. RESULTS with unpaired adenines and cytosines [17], do not seem to react with NiCl, (K. Nejedly, unpublished results) and a procedure for their application at nucleotide sequencing level has been recently elaborated [16]. This procedure is based on increased reactivity of modified bases to subsequent chemical modification prior to performing piperidine cleavage.…”

“…Haloacetaldehydes have been used to probe the conformations of bases in various I&Z junctions (reviewed in [lo]), but with one exception [16] DNA cleavage at the site of modified bases was revealed with Sl nuclease; this enzyme may not, however, recognize and cleave DNA at a single modified base [14]. The single reactive bases that we observe in each strand at the boundaries of the left-handed segment adopted by (dA_dT), sequences (Fig.…”