DNA tertiary structure and changes in DNA supercoiling upon interaction with ethidium bromide and gyrase monitored by UV resonance Raman spectroscopy

Abstract

Abstract The tertiary structure of DNA is important for many of its biological functions. In this work supercoiled and relaxed forms of purified plasmid DNA pBR322 in dilute aqueous solutions are investigated by means of UVRR spectroscopy to assess changes in B‐DNA conformation. Spectral variation in the CO and exocyclic NH 2 vibration above 1600 cm −1 indicate changes in hydrogen bonding. A minor shift of the CN stretching mode of adenosine and guanosine at 1487 cm −1 supports these findings. Changes in ribose conformation are visible in the spectral region 1320–1360 cm −1 by vibrational coupling of the ribose pucker to the vibrations of the purine and pyrimidine bases. The intercalating phenanthridinium drug ethidium bromide is known to reduce the negative supercoiling of DNA. This change in DNA topology is reflected in variations of the UVRR marker bands of DNA identified above. Principal component analysis helped to extract the features of interest from the complex spectra of the intercalation complex. Within the bacterial cells the change in DNA topology is achieved by the action of topoisomerases. In this work, the DNA‐binding subunit GyrA of the enzyme gyrase was extracted from E. coli and applied to relaxed and supercoiled pBR322. The observed changes in the vibrational signature of the relaxed DNA in the presence of GyrA indicate a change of topology towards the supercoiled form. With already supercoiled DNA no further change in DNA topology is observed. Copyright © 2007 John Wiley & Sons, Ltd.