N-Methyl-N-nitrosourea-induced DNA damage detected by the comet assay in Vicia faba nuclei during all interphase stages is not restricted to chromatid aberration hot spots

Abstract

The genotoxic effect of the monofunctional alkylating agent N:-methyl-N:-nitrosourea (MNU) on root-tip nuclei of the field bean, Vicia faba, has been tested by comparative application of three protocols of the comet assay. While the alkaline denaturation/alkaline electrophoresis (A/A) procedure proved to be most sensitive at low doses, the alkaline denaturation/neutral electrophoresis (A/N) procedure yielded an optimal dose-response curve within a wider dose range. With the neutral electrophoresis without alkaline denaturation (N/N) procedure only minimal response was found. MNU-mediated single-strand breaks occurred in nuclei of all interphase stages. Detection of tandemly repeated FOK:I elements on comets by fluorescence in situ hybridization showed an average involvement of these heterochromatin-specific sequences in MNU-mediated single-strand breaks. This, together with previous results, suggests that the pronounced clustering of chromosomal aberrations in heterochromatic regions after treatment with S phase-dependent mutagens is mainly due to an error-prone interference of recombinative repair and replication in damaged basic repeats of large tandem repeat arrays.

DNA damage and repair in Arabidopsis thaliana as measured by the comet assay after treatment with different classes of genotoxins

“…1). The data obtained with both protocols correspond well with data previously obtained for MNU in V. faba nuclei [16]. This confirms that alkylating agents do not directly induce DSBs.…”

“…This confirms that alkylating agents do not directly induce DSBs. Since the increase of the dose-response curve for MNU was significantly higher in the A/A than in the A/N assay [16], we conclude that the A/N protocol detects mainly repair-mediated SSBs while most of the alkali labile sites were detected only with the A/A protocol.…”

RNA-Seq analysis discloses early senescence and nucleolar dysfunction triggered by Tdp1α depletion in Medicago truncatula

“…The frequency of mitosis was calculated over 200 cells, and for each line four replicated samples were analysed. For both DNA diffusion test and single cell gel electrophoresis (SCGE) analysis, nuclei were extracted from leaves as described by Menke et al (2000). Cells undergoing programmed cell death (PCD) or necrosis were distinguished from viable cells using the DNA diffusion test, as indicated by Singh (2000).…”

DNA damage processing and aberration formation in plants

“…Thus, most of the aberrations induced by S phase-dependent clastogens should be derived from DSBs that may result from (rare) positional coincidence of repair-and replication-mediated DNA discontinuities and are preferentially mis-repaired by reciprocal recombination when broken ends involving homologous repeats interact in cis or trans (within a chromatid or between sister or nonsister chromatids) (see Fig. 1 and Schubert et al, 1994;Menke et al, 2000).…”