Clustered DNA lesions Base Excision repair Nucleotide Excision

Clustered DNA lesions • Base Excision repair • Nucleotide Excision repair Base damage Clustered DNA lesion Double strand break Single strand break • Homologous recombination Base Excision repair • Non-Homologues end joining

Investigation goal Visualization and analysis of the structure of clustered DNA double -strand breaks (DSBs) in human fibroblasts irradiated with 15 N ions.

Experimental scheme Parameters of irradiation Irradiation of human fibroblasts with 15 N and 60 Co γ-rays Fixation of cells at different times post-irradiation Type of irradiation LET, ke. V/µm Dose, Gy Radiation source 15 N 181. 4 13 1. 25 «Genome-M» Setup set up of U -400 M cyclotron γ-rays 0. 3 - 1 «Rocus-M» Immunostaining procedure Fluorescent microscopy Visualization of induced DSBs and base damage Energy, Me. V/n Secondary antibody conjugated to fluorescent label Primary antibody OGG 1 53 BP 1 Quantitative and qualitative analysis of the obtained results DNA DSB Base damage Clustered DNA DSBs

Analysis the of the 53 BP 1/OGG 1 clusters structure 30 20 10 0 1 2 3 4 5 6 7 8 40 30 20 10 0 9 10 11 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 The number of OGG 1 foci in clusters (n) Number of clusters with n OGG 1 foci, % 48. 3% 50 2 3 4 5 6 7 8 9 40 30 20 10 11 60 60. 2% 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 The number of OGG 1 foci in clusters (n) 53 BP 1 38. 2% 50 60 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 60 62. 6% 50 40 30 20 10 0 2 3 4 5 6 7 8 9 10 11 The number of OGG 1 foci in clusters (n) OGG 1 1 2 3 4 5 6 7 8 9 10 11 The number of 53 ВР 1 foci in clusters (n) 60 65% 50 40 30 20 10 0 1 45. 7% 50 The number of 53 ВР 1 foci in clusters (n) 60 1 60 Number of clusters with n 53 BP 1 foci, % 40 36. 5% 50 24 h Number of clusters with n OGG 1 foci, % 20. 9% 50 60 Number of clusters with n OGG 1 foci, % 60 4 h Number of clusters with n 53 BP 1 foci, % 1 h Number of clusters with n 53 BP 1 foci, % 15 min 1 2 3 4 5 6 7 8 9 10 11 The number of OGG 1 foci in clusters (n)

Conclusions • Qualitative analysis of the obtained images of stained human fibroblasts nuclei showed that accelerated 15 N ions induce more repair resistant clustered DNA DSB with highorder structure compared to γ-rays. • In general, the analysis of the structure of 53 BP 1/OGG 1 clusters revealed that the number of OGG 1 foci is greater than 53 BP 1 foci per cluster. The results may demonstrate that there are several base damage to one DSB. • It was revealed the number of 53 BP 1 and OGG 1 foci per cell decreased but the most high-order OGG 1/53 BP 1 clusters were observed at 24 h after irradiation. The result may indicate the difficulty in the repair processes of all types of DNA lesions included in clusters.

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