Journal of Chromatography B, 741 (2000) 243–255 www.elsevier.com / locate / chromb Analysis of gamma radiation-induced damage to plasmid DNA using dynamic size-sieving capillary electrophoresis a ,1 a, b ,2 * Stacey A. Nevins , Barbara A. Siles , Zeena E. Nackerdien a Department of Chemistry, The College of William and Mary, P .O. Box 8795, Williamsburg, VA 23187-8795, USA b Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Received 24 September 1999; received in revised form 25 January 2000; accepted 31 January 2000 Abstract Bacterial plasmids and the chromosomal DNA of many organisms adopt naturally the negatively supercoiled conforma- tion. Therefore, the irradiation of such plasmids could be used to model conformational changes of chromosomal DNA associated with externally-induced damage. We have applied dynamic size-sieving capillary electrophoresis (CE) to monitor the damage of three DNA plasmids, over an unprecedented base pair (bp) size range (2870–27 500 bp), upon exposure to g-radiation (20–400 Gy). Predominantly, CE with UV absorbance detection in the absence of DNA intercalating dyes was employed to preclude undesirable, induced plasmid conformational changes. Plasmid samples and their enzymatic digestion products were analyzed using both CE and slab gel electrophoresis (SGE) in order to verify the conformation of sample components. Relative to SGE, CE analyses revealed more fine structural features of plasmid degradation.  2000 Elsevier Science B.V. All rights reserved. Keywords: Plasmids; Gamma-radiation; DNA 1. Introduction antibiotics, heavy metals or UV radiation. In the cell, bacterial plasmids adopt a negatively supercoiled Plasmids are naturally occurring, circular double- conformation. Large plasmids and chromosomal stranded DNA (dsDNA) molecules that replicate DNA are organized into discrete topological domains independently of the genome. Many plasmids encode with defined numbers of supercoils and helical turns. unique metabolic functions such as resistance to These supercoils are critical to the regulation of several biological processes, including DNA replica- tion and gene expression [1]. Other possible plasmid conformations include the open circular conformer *Corresponding author. The University of Denver Research (either the relaxed form or the nicked form) and the Institute, 2050 E. Iliff Ave., Denver, CO 80208, USA. Tel.: 11-303-8717-464, fax: 11-303-8714-119. linear conformer. The nicked open circular form is E-mail address: bsiles@du.edu (B.A. Siles) generated by a single-stranded break (a nick), which 1 Present address: Department of Chemistry, The University of allows supercoiled DNA to unwind [1,2]. Breaks Michigan, 930 N. University, Ann Arbor, MI 48109-1055, USA. 2 may be induced by (i) brief treatment with restriction Present address: Qualus, 420 East 51 Street, Suite A, New endonucleases, (ii) radical processes mediated by York, NY 10022, USA. Tel.: 11-212-2230-813; fax: 11-212- 2230-893. reactive oxygen species, (iii) ionizing radiation, or 0378-4347 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0378-4347(00)00106-7