Formation of 53BP1 foci and ATM activation under oxidative stress is facilitated by RNA:DNA hybrids and loss of ATM-53BP1 expression promotes photoreceptor cell survival in mice

Abstract

Background: Photoreceptors, light-sensing neurons in retina, are central to vision. Photoreceptor cell death (PCD) is observed in most inherited and acquired retinal dystrophies. But the underlying molecular mechanism of PCD is unclear. Photoreceptors are sturdy neurons that survive high oxidative and phototoxic stress, which are known threats to genome stability. Unexpectedly, DNA damage response in mice photoreceptors is compromised; mainly due to loss of crucial DNA repair proteins, ATM and 53BP1. We tried to understand the molecular function of ATM and 53BP1 in response to oxidative stress and how suppression of DNA repair response in mice retina affect photoreceptor cell survival. Methods: We use the state of art cell biology methods and structure-function analysis of mice retina. RNA:DNA hybrids (S9.6 antibody and Hybrid-binding domain of RNaseH1) and DNA repair foci (gH2AX and 53BP1) are quantified by confocal microscopy, in retinal sections and cultured cell lines. Oxidative stress, DNA double strand break, RNaseH1 expression and small-molecule kinase-inhibitors were used to understand the role of ATM and RNA:DNA hybrids in DNA repair. Lastly, retinal structure and function of ATM deficient mice, in Retinal degeneration 1 (Pde6brd1) background, is studied using Immunohistochemistry and Electroretinography. Results: Our work has three novel findings: firstly, both human and mice photoreceptor cells specifically accumulate RNA:DNA hybrids, a structure formed by re-hybridization of nascent RNA with template DNA during transcription. Secondly, RNA:DNA-hybrids promote ataxia-telangiectasia mutated (ATM) activation during oxidative stress and 53BP1-foci formation during downstream DNA repair process. Thirdly, loss of ATM -in murine photoreceptors- protract DNA repair but also promote their survival. Conclusions: We propose that due to high oxidative stress and accumulation of RNA:DNA-hybrids in photoreceptors, expression of ATM is tightly regulated to prevent PCD. Inefficient regulation of ATM expression could be central to PCD and inhibition of ATM-activation could suppress PCD in retinal dystrophy patients.

Calcitriol Prevents RAD51 Loss and cGAS‐STING‐IFN Response Triggered by Progerin

“…Our previous studies in primary fibroblasts subjected to a long protocol of H 2 O 2 treatment that causes oxidative stress‐induced senescence revealed a marked downregulation of RAD51 . However, acute treatment with H 2 O 2 at a concentration that causes oxidative stress and DNA damage but not cell senescence or death results in no changes in RAD51 levels in either control or progerin‐expressing cells (Figure 3C). This treatment with H 2 O 2 did increase the levels of 53BP1 nuclear foci, consistent with accumulation of DNA damage (Figure 3D).…”

“…Calcitriol (1α,25‐dihydroxyvitamin D 3 ), the hormonal active form of vitamin D, was added to cells either 2 days prior or together with doxycycline at a concentration of 10 −7 m . To induce oxidative stress, HDFs that had been growing in media containing doxycycline or vehicle for 2 days were exposed to 100 µ m H 2 O 2 for 2 h. This treatment was shown to induce oxidative stress …”

Differences in the Response to DNA Double-Strand Breaks between Rod Photoreceptors of Rodents, Pigs, and Humans

“…We and others have previously shown that fully differentiated rod PRs of adult mice repair DSBs very inefficiently [8,9]. Of note, these cells fail to efficiently express the DNA damage response kinase ataxia-telangiectasia mutated (ATM), although an alternative splicing form seems to exist [9]. In addition, the DSB-induced ATM autophosphorylation is strongly impaired in adult mouse PRs, suggesting a strong diminution of ATM kinase activity [8].…”

“…In order to improve the HR efficiency in postmitotic cells, detailed knowledge about the particularities of individual cell types with regard to their DNA damage response and their DSB repair capacities is of great importance. We and others have previously shown that fully differentiated rod PRs of adult mice repair DSBs very inefficiently [8,9]. Of note, these cells fail to efficiently express the DNA damage response kinase ataxia-telangiectasia mutated (ATM), although an alternative splicing form seems to exist [9].…”

“…Although inverted rod nuclei remain Cells 2020, 9, 947 3 of 16 overtly functional [17], they exhibit an altered epigenetic landscape [22] and a changed expression of some common proteins [23]. This might explain the downregulation of ATM and KAP1, which in turn causes defects in DNA damage signaling and DSB repair in adult rods [8,9]. This is supported by the fact that undifferentiated rod progenitors (e.g., at P4), which still possess conventional nuclei, have a normal DSB repair efficiency [8].…”