Abstract
Replication errors that are caused by mutagens are critical for living cells. The aim of the study was to analyze the distribution of a DNA replication pattern on chromosomes of the H. vulgare ‘Start’ variety using pulse 5-ethynyl-2′-deoxyuridine (EdU) labeling, as well as its relationship to the DNA damage that is induced by mutagenic treatment with maleic hydrazide (MH) and γ ray. To the best of our knowledge, this is the first example of a study of the effects of mutagens on the DNA replication pattern in chromosomes, as well as the first to use EdU labeling for these purposes. The duration of the cell cycle of the Hordeum vulgare ‘Start’ variety was estimated for the first time, as well as the influence of MH and γ ray on it. The distribution of the signals of DNA replication along the chromosomes revealed relationships between DNA replication, the chromatin structure, and DNA damage. MH has a stronger impact on replication than γ ray. Application of EdU seems to be promising for precise analyses of cell cycle disturbances in the future, especially in plant species with small genomes.
Highlights
Data regarding the effects of mutagens on plant nuclear genomes and DNA replication are of great importance
We present the distribution of the DNA replication pattern on chromosomes using pulse ethynyl-2 -deoxyuridine (EdU) labeling and analyze its relationship with the DNA damage that is induced by mutagenic treatment with maleic hydrazide (MH) and γ ray
Such a high resolution of the EdU method indicates that it can be used for more precise analyses of cell cycle disturbances, for plant species with large chromosomes, but especially for those with small chromosomes
Summary
Data regarding the effects of mutagens on plant nuclear genomes and DNA replication are of great importance. The spatiotemporal patterns of DNA replication in nuclei were recently characterized in detail in control cells [1], as well as in relation to DNA damage and mutagenesis [2] using a quantitative analysis. To date there is no similar data on the effects of mutagens on the pattern of DNA replication on chromosomes. One of the disadvantages of using BrdU is degradation of the chromatin structure during denaturation step, which is especially inconvenient in the context of an analysis of DNA damage during mutagenesis. The relatively large size of the detection sites caused by the need to use specific antibodies to detect BrdU is an unfavorable feature of an analysis of DNA replication sites, especially in the case of an analysis of the signals in chromosomes. Its good preservation of chromatin and high resolution make this technique useful in a detailed analysis of the effects of mutagens on the S-phase [2]
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