Invited Speaker
Lynn Zechiedrich, (www.bcm.edu/labs/zechiedrich/?PMID=1623), Baylor College of Medicine at Texas, USA.
Supercoiled minicircle DNA: a superior substrate for the study of DNA structure and topoisomerase-DNA interactions
The study of topoisomerases has been hampered by a lack of manipulable supercoiled DNA substrates. We developed a protocol to produce milligrams of supercoiled minicircle DNA, (<500 bp), with well-defined linking number (Lk). We isolate individual topoisomers over a wide range of positive and negative Lk. We have studied the partitioning of Lk into twist and writhe. Many models of DNA elasticity assume that positively supercoiled DNA is equal and opposite to negatively supercoiled DNA. We show, however, that positively supercoiled minicircles have a much higher propensity to writhe. Equilibrium and competition gel-shift assays revealed that human topoisomerase IIα binds to supercoiled minicircle DNA two orders of magnitude more tightly than to linear or relaxed substrates. This tight binding is concomitant with the onset of writhing, suggesting that type II topoisomerases recognize structural features that are characteristic of writhe. Positively and negatively supercoiled minicircles were bound with equal affinity indicating that the reported preferential relaxation of positively supercoiled DNA is not manifested at the initial binding and recognition step.