Differential Recruitment of DNA Repair Proteins KU70/80 and RAD51 upon Microbeam Irradiation with α-Particles

Abstract

In addition to representing a significant part of the natural background radiation exposure, α-particles are thought to be a powerful tool for targeted radiotherapy treatments. Understanding the molecular mechanisms of recognition, signaling, and repair of α-particle-induced DNA damage is not only important in assessing the risk associated with human exposure, but can also potentially help in identifying ways of improving the efficacy of radiation treatment. α-particles (He2+ ions), as well as other types of ionizing radiation, and can cause a wide variety of DNA lesions, including DNA double-strand breaks (DSBs). In mammalian cells, DNA DSBs can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). Here, we investigated their dynamics in mouse NIH-3T3 cells through the recruitment of key proteins, such as the KU heterodimer for NHEJ and RAD51 for HR upon localized α-particle irradiation. To deliver α-particles, we used the MIRCOM microbeam, which allows targeting of subnuclear structures with submicron accuracy. Using mouse NIH-3T3 cells, we found that the KU heterodimer is recruited much earlier at DNA damage sites marked by H2AX phosphorylation than RAD51. We also observed that the difference in the response of the KU complex and RAD51 is not only in terms of time, but also in function of the chromatin nature. The use of a microbeam such as MIRCOM, represents a powerful tool to study more precisely the cellular response to ionizing irradiation in a spatiotemporal fashion at the molecular level.

Decline of DNA damage response along with myogenic differentiation

“…Briefly, after damage induction, the accumulation of FEN1 correlates proportionally with the substrate produced by the BER reaction and the disappearance of the FEN1-YFP signal correlates with the disappearance of the substrate coinciding with the end of BER. We induced local oxidative damage (LOD), within nuclei of myoblasts and myotubes, with different damaging techniques that have been previously shown to produce different kinds of DNA lesions (Mari et al, 2006;Bobyk et al, 2022). We observed a clear reduction of the accumulation of FEN1 in LOD in myotubes versus myoblasts, and we could show that the turnover rate of FEN1 on LOD in myotubes is very slow compared with the one measured in myoblasts and previously measured in MEFs (Kleppa et al, 2012).…”

“…Irradiation with α-particles is known to induce, in addition to DNA strand breaks, oxidative base lesions (Danforth et al, 2022). We thus performed locally irradiation within cell nuclei with a predetermined number of 6 MeV α-particles with a micrometric spatial resolution (Bobyk et al, 2022;Vianna et al, 2022). We measured the BER activity in mononuclear cells including MB and myocytes (MB + MC), which are mononuclear myogenic cells, and in MT, upon local irradiation with 1,000 α-particles (Fig 1C and D).…”

“…The beam is focused down to a few micrometers by a quadruplet of magnetic quadrupoles and extracted in air through a thin silicon nitride membrane (150 nm thick, 1 × 1 mm 2 ; Silson Ltd). It is then sent to the targeted zone by electrostatic scanning plates for a given number of ions or for a given amount of time, as previously described (Bobyk et al, 2022;Vianna et al, 2022). The low energy α-particles have low penetration capacity and thus short travel distance through matter, therefore the cells are seeded in a special cell dish with a 4-µm thick polypropylene foil (Goodfellow) (Bourret et al, 2014).…”

“…The low energy α-particles have low penetration capacity and thus short travel distance through matter, therefore the cells are seeded in a special cell dish with a 4-µm thick polypropylene foil (Goodfellow) (Bourret et al, 2014). The LET of the α-particles after going through the extraction window (150 nm), a residual air layer (250 μm), and the polypropylene foil (4 μm) is 84 keV/μm (Bobyk et al, 2022). To provide optimal cell growth conditions, the polypropylene foil is pre-coated with 10 ng/μl of Cell-Tak (Corning, Thermo Fisher Scientific) followed by Matrigel (Corning, Thermo Fisher Scientific).…”

miR-302d Targeting of CDKN1A Regulates DNA Damage and Steroid Hormone Secretion in Bovine Cumulus Cells

“…In addition (Figure 4), the expression of CDKN1A in bovine cumulus cells transfected with miR-302d mimics decreased, the rate of γH2AX-positive cells decreased significantly, the cell cycle changed, proliferation was promoted, apoptosis was inhibited, and the secretion levels of E 2 and P 4 were increased. RAD51 can regulate the DDR through multiple pathways [62,63]. The expression of several targeted genes is controlled by a single miRNA [64].…”