Studies on DNA supercoiling in eukaryotic cells
Creators: name: Sandhu, Laurie Cochran
Publisher: name: University of Utah
Year: 2012
Subjects: Tumor Cells
Topoisomerases

description: The state of DNA supercoiling has been postulated to be an important regulator of many cellular functions, including replication, transcription, recombination, and repair. A difficulty in the study of the coiling state of high molecular weight eukaryotic DNA, however, is the fact that few assays exist to determine the topology of high molecular weight DNA. Fluorescent DNA stains, which exhibit different spectral properties with DNA in defined topological states, might provide an assay system for high molecular weight DNA. In the present studies, the vital fluorescent dye, Hoechst 33342 (HO 33342) was used to study the plasmid pBR322 in defined topological states. Supercoiled (Form I) or relaxed covalently closed circular (Form JO) pBR322 was probed with HO 33342 and the fluorescence of the dye-DNA complexes was monitored on a spectrofluorometer. It was found that HO 33342, independent of both the dye and DNA concentration, exhibited a 30% greater fluorescence with Form I than with Form 1° DNA. However, when the two forms were mixed in various proportions and the fluorescence of HO 33342 monitored, it was found that while the fluorescence increased qualitatively with the amount of Form I present in the mixture, a consistent quantitative relationship was not obtained. These results suggest that the HO 33342 fluorescence assay is not appropriate as a quantitative measure of the proportions of various topological states of DNA.  A biophysical assay which has been used for studying the state of supercoiling of high molecular weight DNA is the nucleoid sedimentation assay. Nucleoids sediment in neutral sucrose gradients containing various concentrations of intercalators in a manner which suggests that the DNA is organized into domains of supercoiling. The nucleoid sedimentation assay was used to investigate the effect of damage and repair of supercoiling after ionizing radiation in murine L929 cells. A dose of 10 Gy X-rays was found to reduce nucleoid sedimentation to 60% of the unirradiated value, and a repair period at 370 resulted in a nucleoid structure with increased sedimentation, suggesting that the damage to DNA supercoiling was repairable. Agents which might have been expected to interfere with the repair of damage to DNA supercoiling, such as hyperthermia and treatment with novobiocin, a gyrase inhibitor, proved ineffective in inhibiting the restoration of nucleoid compaction.  If, as has been suggested, the state of DNA supercoiling regulates DNA replication and gene expression, then an appropriate biological test system for this hypothesis would be a cell line in which DNA replication is inhibited and possibly, altered gene expression exists. The murine mammary tumor cell line, designated 67, which can be obtained in two distinct growth states, designated P and Q for proliferating and quiescent, respectively, was employed in nucleoid sedimentation studies. Two probes of supercoiling state were used; ionizing radiation, which relaxes supercoils and ethidium bromide, which relaxes and then rewinds supercoils. It was found that the nucleoid DNA isolated from Q cells behaved as if it were not under torsional strain. Nucleoids isolated from 0 cells, in contrast to those isolated from P cells, did not show a decrease in sedimentation after ionizing radiation and did not exhibit the classic biphasic sedimentation response to increasing concentrations of ethidium bromide. As it is known that DNA replication is inhibited in 670 cells and it is possible that only those genes necessary for cell survival are being transcribed, this finding may provide a biological correlate between the state of DNA supercoiling and the growth state of eukaryotic cells.