DNA loop domain organization in nucleoids from cells of different types.

The loop domain organization of chromatin plays an important role in transcription regulation and thus may be assumed to vary in cells of different types. We investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) for nucleoids obtained from human lymphocytes, lymphoblasts and glioblastoma T98G cells. The results confirm our previous observation that there are three parts of DNA in nucleoids: DNA on the nucleoid surface, loops up to ∼150 kb inside the nucleoid, and larger loops that cannot migrate. However, the relative amounts of the three parts were found to be very different for different cell types. The distributions of the loop length up to 150 kb were shown to be exponential, with the distribution parameter, the loop density, to be dependent on the cell type.

Single nucleus versus single-cell gel electrophoresis: kinetics of DNA track formation.

Single-cell gel electrophoresis, or the comet assay, is usually performed with nucleoids prepared after a lysis of either whole cells (more often) or isolated cell nuclei (rarely). Electrophoretic properties of the second type of nucleoids have never been investigated carefully. We measured the kinetics of the DNA exit from nuclei-derived nucleoids in comparison with cell-derived nucleoids. The results show that general organization of the nuclei-derived nucleoids is not changed very much in comparison with nucleoids commonly obtained from whole cells. At the same time, in contrast to the cell-derived nucleoids, for which the exit is stepwise and cooperative, the DNA exit from the nuclei-derived nucleoids can be described by a simple monomolecular kinetics. This difference is probably due to agarose penetration into nuclei (but not into cells) before polymerization of the agarose gel. We suggest that single-nucleus gel electrophoresis may be a way for the comet assay standardization.

DNA loop domain organization as revealed by single-cell gel electrophoresis.

At higher order levels chromatin is organized into loops. This looping, which plays an important role in transcription regulation and other processes, remains poorly understood. We investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay). The migration of a part of the loops was shown to be reversible after switching off electrophoresis and to be sensitive to intercalation-induced changes in supercoiling. Another group of the loops migrates rapidly, the rate being insensitive to the supercoiling level. The largest part of the loops cannot migrate at all, presumably because of their large size. The loop ends can be detached in the presence of high concentrations of intercalators or protein denaturants, thus increasing the fraction of DNA that cannot migrate in the gel. The distribution of the loop length up to 100kilobases appears to be consistent with the fractal globule organization.

Kinetics of comet formation in single-cell gel electrophoresis: loops and fragments.

We investigated the mechanisms of DNA exit during single-cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single-strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.

Physical principles and new applications of comet assay.

The comet assay is a sensitive method to assess DNA damages in single cells. The approach consists of an analysis of electrophoretic migration of DNA from nucleoids obtained after cell lysis in a thin layer of agarose. Although the method is widely used the physical mechanisms of DNA track formation remained to be rather elusive for a long time. This review is devoted to our recent results pertaining to this subject, using an original approach based on the kinetic measurements of the comet formation. We argue that linear DNA fragments give an essential contribution into the tail formation in the alkaline conditions and, at neutral pH, when the level of DNA damages is very high. On the other hand, in the neutral comet assay at low levels of DNA damages (and also in the case of undamaged cells) the tail is formed by extended DNA loops. These loops are about the same as chromatin loops in the cell nuclei. Kinetic measurements in the comet assay give an opportunity to investigate the topology of the loops and large-scale features of the loop domain organization (and re-organization) in nucleoids obtained from different cell types.