Superhelix Organization by DNA Curvature as Measured Through Site-specific Labeling Claudia Pfannschmidt and Jo È rg Langowski* Division Biophysics of Macromolecules, German Cancer Research Center Im Neuenheimer Feld 280 D-69120 Heidelberg, Germany For determining the position of a de®ned site in a superhelical DNA we have developed a method for introducing a covalent biotin label at a speci®c sequence while preserving the superhelicity. This is done by ®rst introducing a speci®c nick, labeling the DNA by limited nick translation and sealing the nick with ligase. The superhelicity is controlled by includ- ing ethidium in the ligation reaction. Using scanning force of microscopy on DNAs labeled by this method, we have then compared the position of streptavidin markers at a speci®c site relative to the end loop of the superhelix. We found that in DNAs with permanently curved inserts the label is located preferentially at a de®ned distance from the end loop, while in controls without curved inserts the label position was random. This indicates that curves are located in or near the end loops in a superhelix. # 1998 Academic Press Limited Keywords: superhelical DNA; EcoRI-nicking; biotin label; streptavidin- POD; scanning force microscopy *Corresponding author Introduction In the regulation of transcription long-range ordering in DNA plays an important role: tran- scription factors bound to enhancers contact the transcription machinery at the promoter by loop- ing of the intervening DNA (von Hippel et al., 1996). The effectiveness of loop formation depends on the ¯exibility and conformation of the DNA. One possible mechanism to enable interactions over a distance is the formation of a superhelix that brings distant DNA regions into close proxi- mity. The global structure of a superhelix is in¯u- enced by local bending caused by proteins or intrinsic curvature (Klenin et al., 1995; Kremer et al., 1993; Laundon & Grif®th, 1988). Although many hypotheses exist about the role of local DNA struc- tural changes in determining superhelical DNA conformation, experimental approaches have been rare because of the elusiveness of the material: large DNAs are ¯exible molecules whose shape ¯uctuates and depends strongly on experimental conditions. One way of monitoring the confor- mational changes of superhelical DNA is to have markers at speci®c sites from which one can detect the position of a segment of a long DNA relative to the rest of the molecule. The purpose of this work was to quantify the orientation of a superhelical DNA by a curved insert. To this aim, we introduced a site-speci®c label into superhelical DNA whose position in the superhelix could be directly observed by scanning force microscopy (SFM). Introducing a site-speci®c label into superhelical DNA requires sequence-speci®c recognition by the labeling reagent. We have shown in an earlier work (Pfannschmidt et al., 1996) that superhelical DNA can be labeled by triplex-forming oligonu- cleotides (TFOs). The TFOs carry a biotin label on their 3 0 end and a psoralen moiety on their 5 0 end that under UV irradiation crosslinks with the DNA, thus keeping the label attached even when buffer conditions are changed. Although the TFO strategy works well it might be a disadvantage for some physical measurements that a rather large group (a 20 to 30mer oligonucleotide coupled to psoralen and biotin and a streptavidin conjugate bound to the biotin) is attached to the DNA. Fur- thermore, a rather long sequence is necessary to achieve speci®c recognition. We have therefore developed an alternative labeling method based on speci®c nicking and nick translation with Escherichia coli DNA polymerase I. With this method, biotin-labeled dUTP is directly Abbreviations used: SFM, scanning force microscopy; TFO, triplex-forming oligonucleotide; strept-POD, streptavidin horseradish peroxidase. J. Mol. Biol. (1998) 275, 601±611 0022±2836/98/040601±11 $25.00/0/mb971476 # 1998 Academic Press Limited