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Analysis of Repaired D. radiodurans DNA by 5-BrdU Density Labeling Post-Irradiation

This study investigates the DNA repair mechanisms in D. radiodurans through the use of 5-BrdU density labeling. The cells were subjected to irradiation followed by the addition of 5-BrdU, allowing for a detailed analysis of DNA density profiles via CsCl equilibrium density centrifugation. Single-stranded and double-stranded DNA molecules were observed to assess the integrity and repair efficiency of genomic DNA. The findings illustrate distinct patterns in pre- and post-labeled DNA in native and denatured states, enhancing our understanding of D. radiodurans's resilience to DNA damage.

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Analysis of Repaired D. radiodurans DNA by 5-BrdU Density Labeling Post-Irradiation

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  1. HH HL LL HH HL LL HH HL LL c b a H L H L H L c1 b1 a1 radman_figS1 Figure S1. Analysis of repaired D. radiodurans DNA by 5-BrdU density labelling.D. radiodurans thy- cells were radioactively and density labelled by the growth in the presence of 3H-thymidine and 5-BrdU, respectively. Density labelling was performed by adding 5-BrdU to the medium only after irradiation. Genomic DNA was extracted and analysed by CsCl equilibrium density centrifugation. HH, HL and LL refer to double-stranded heavy/heavy, heavy/light and light/light molecules. H and L refer to single-stranded heavy and light molecules. a, a1, 3H-thymidine «pre-labelled» DNA (labelled before irradiation). b, b1, 3H-thymidine «post-labelled» DNA (labelled after irradiation). c, c1, 3H-thymidine «pre- and post-labelled» DNA. a, b, c, Native DNA in neutral CsCl gradients. a1, b1, c1,Denatured DNA in alkaline CsCl gradients. Open black circles: DNA density profile from unirradiated cell culture grown for 2.5 h (one mass-doubling) in 5-BrdU-supplemented medium. Closed red circles: DNA density profile from 7 kGy-irradiated cells grown for 3 h in 5-BrdU-supplemented medium.

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