How does radiation quality affect induction and repair

How does radiation quality affect induction and repair of DNA double-strand breaks? Ježková Lucie «Alushta-2017» 12 -19 June 2017

Ionizing radiation IR 1 unit of the dose Sparsely ionizing (low-LET; γ-rays, X-rays) Densely ionizing (high-LET, charged particles) Low-LET Isolated DNA lesions Low-LET • DSB – the most serious Clustered DNA lesions + IR • the most severe damage • 2 or more isolated lesions in close proximity High-LET ionization 20 Å High-LET DNA leasion • incidence grows with increasing LET • slower repair or non-repairable

Experimental • Cell culture of human skin fibroblasts • Irradiation: γ-rays – 1 Gy 10 ions – 3 particles/nucleus 0 Irradiation γ- rays (60 Со) 11 B ions 20 Ne ions LET, ke. V/μm Energy, Me. V/n Dose, Gy 0, 3 - 1 135 8, 3 1 129 47, 5 1. 2 • visualization of DSBs in situ - co-immunostaining of γH 2 AX and 53 BP 1 proteins (specific antibodies labelled with a fluorochrome used to visualize particular proteins in spatially fixed nuclei) „Genome-M“, U-400 M

Aims WHY? ? Morphology evaluation 1. Size of γH 2 AX/53 BP 1 foci 2. Shape of γH 2 AX/53 BP 1 3. foci (circularity) Complexity of γH 2 AX/53 BP 1 foci

γH 2 AX/53 BP 1 foci; μm 2 γH 2 AX/53 BP 1 foci “Circularity”: π×(area/perimeter 2) Area 4 h after 1 Gy 11 Birradiation Circularity γH 2 AX/53 BP 1 foci area and circularity Time after irradiation, h

γH 2 AX/53 BP 1 foci complexity 1 • Presence of γH 2 AX/53 BP 1 foci clusters γH 2 AX/53 BP 1 γH 2 AX 3 individual foci 1 γH 2 AX/53 BP 1 cluster x-y 2 D x-z x-z 3 D x-z 2 individual foci

γH 2 AX/53 BP 1 foci complexity 2 1 h 15 min 9% γ-rays 11 B 14 % 1% 19 % 81 % 0, 3 % 75 % 27 % 58 % 5% 20 Ne 64 % 25 % 2% 84 % 90 % 29 % 6% 1% 30 % 44 % 18 % 1% 1% 24 h 4 h 6% 37 % 42 % 10 % 2% 3% 1% 1% 4% 6% 2% 29 % 11 % 0, 3 % 87 % 46 % 13 % 12 22 % % 28 % 4% 6% 4% 24 % 68 % 8% 6% 2% 28 % 19 % 2% 77 % 33 % 11 % 96 h 26 % 13 23 % % 56 % 35 % 3% 6% Cluster complexity 1 focus 2 foci 3 foci 4 foci 5 foci 6 foci 7 foci 8 foci • γ-rays – mostly individual foci; clusters composed mainly of 2 foci • 11 B ions – more complex clusters composed up to 6 foci; greatest complexity at 4 and 24 h • 20 Ne ions – the highest complexity; clusters composed up to 8 individual smaller foci

Conclusions q High-LET ions evoked formation of larger γH 2 AX/53 BP 1 foci which were more shape irregular compared to those induced by low-LET γ-rays § The largest foci were formed at 24 h after irradiation with neon ions. q Observed presence of γH 2 AX/53 BP 1 foci clusters especially after irradiation with accelerated ions § The highest complexity and portion of γH 2 AX/53 BP 1 foci clusters was observed after neon irradiation, where up to 8 smaller foci was detected. q The complexity of γH 2 AX/53 BP 1 foci clusters correlated with the size and circularity analysis Induction of the most complex, clustered DNA damage by NEON ions → thus the slowest kinetics of DSB repair and fastest DSB recognition They have similar LET valus so WHY? ? ?

Track structure: characteristic spatial pattern of energy deposition Composition of particle tracks depends on: ● traversed material ● type of the particle and its charge (Z) ● kinetic energy (E) 11 B 20 Ne 2 nm. ZE == 58, 3 Me. V/n Z = 10 E = 46, 8 Me. V/n LET = 132 ke. V/μm LET = 135 ke. V/μm RITRACKS v. 3. 1, NASA/Johnson Space Center Element 20 Ne 11 B Track core radius Rc, nm 3, 0 1, 2 δ-rays radius Rp, µm 15, 6 1, 6 Calculations from A. Chatterjee: Chatterjee & Holley (1993)

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DNA in nucleus Levels of chromatin packing DNA double helix 2 meters Different chromatin domains with distinct structure and function „ beads-on-a-ring“ form of chromatin 30 -nm chromatin fiber of packed nucleosomes section of chromosome in an extended form looped domains Falk et al, BBA Mol Cell Biol 2008 protein scaffold condensed section of chromosom Clustered DNA damage!!!