Chemical probing of the homopurine·homopyrimidine tract in supercoiled DNA at single‐nucleotide resolution

Abstract

Local structure of the homopurine homopyrimidine tract in a supercoiled plasmid pEJ4 was studied using chemical probes at single-nucleotide resolution. The conformation of the homopyrimidine strand was probed by osmium tetroxide, pyridine (Os,py) while that of the homopurine strand was tested by diethyl pyrocarbonate (DEPC), i.e. by probes reacting preferentially with single-stranded DNA. At weakly acidic pH values, a strong Os,py attack on three nucleotides at the centre of the (dC-dT),, block and a weaker attack on two nucleotides at the end of the block were observed. DEPC modified adenines in the S-half of the homopurine strand. Os,py modification at the centre of the block corresponded to the loop of the hairpin formed by the homopyrimidine tract, while DEPC modification corresponded to the unstructured half of the homopurine strand in the model of protonated triplex H form of DNA.

Protonated triplex DNA in E. coli cells as detected by chemical probing

“…We have shown [5,7] that osmium tetroxide, pyridine site-specifically modifies the centre of the homopyrimidine strand of the supercoiled pEJ4 DNA in vitro at acidic pH values up to pH 6.0 while no such modification occurs at pH 7.8. The presence of the triplex H-DNA in pEJ4 in vitro is manifested by a strong modification in the centre of the (dC-dT)n sequence and a weaker modification at the triplex boundary [7].…”

“…Chemical probing of some polypurine-polypyrimidine tracts in recombinant plasmids yielded direct and compelling evidence [5][6][7][8][9][10][11] in favour of the model of intramolecular protonated triplex H-DNA [1][2][3][4] proposed by Lyamichev et al H triplex is stabilized in vitro by negative supercoiling and acidic pH; at neutral pH under specific conditions [9,13] a different type of triplexes was observed. H triplex introduces a curvature [8,12] or a hinge into the DNA molecule.…”

“…The cells were then treated with 2 mM Os,bipy at 37°C for 30 rain (if not stated otherwise), washed and DNA was isolated [14] by the boiling method. DNA Os,bipy modification in vitro [5,7] and in situ [14] was performed as described, pL153 DNA was cleaved with Tthllll or AspI, labelled with [a-32p]dTTP using the Klenow fragment and treated according to the standard protocol [20]. Intracellular pH measurement: cells were incubated with 100/zM fluorescein diacetate and the fluorescence intensity at 520 nm after excitation at 435 and 490 nm was recorded with a Perkin-Elmer MPF 3 spectrofluorimeter as described [21].…”

Osmium tetroxide, N,N,N',N'‐tetramethylethylendiamine A new probe of DNA structure in the cell

“…Osmium tetroxide probes of DNA structure have a number of advantages as compared to enzymatic and other chemical probes [4,5,16]; among these is the established chemistry of their interaction with DNA components (for review, see [18]), detection of the osmium binding sites at single nucleotide resolution [5,[19][20][21][22][23], availability of antibodies against osmiummodified DNAs [5,24], etc. Os,TMEN represents a second osmium probe which can be applied to probe DNA structure in situ.…”

“…KMnO4 oxidizes DNA bases reacting mainly with thymine and to a much lesser extent with cytosine and guanine, i.e., its reactivity towards bases is similar to that of osmium tetroxide itself [18] and to some extent also to that of Os,bipy [5,26]. A combination of chemical probes in the studies of the DNA structure in vitro has been very useful [17,19,22,27] and one can expect that it will be even more important in the studies of the DNA structure in the cell mainly due to the complexity of the environment inside the cell.…”