Rad51 Suppresses Innate Immune Response By Blocking Mre11-Mediated Degradation Of Newly Replicated Genome

Purpose of the study: Eukaryotic cells accrue DNA damage as a result of endogenous metabolic activities such as DNA replication, recombination errors or environmental exposures such as ionizing radiation, ultra-violet light and chemical mutagens. Unrepaired DNA damage leads to tumorigenesis. Rad51 is a multifunctional protein that plays a central role in DNA replication and homologous recombination repair. It is known that defects in Rad51 function can cause cancer. The goal of this study is to identify a novel role for Rad51 outside of its known functions in DSB repair and replication fork processing. Methods: Since Rad51 knockout is lethal to cells, we generated an inducible system in which we can down-regulate Rad51 expression in HT1080 cells after Doxycycline treatment. To determine the effect of Rad51-knockdown in global gene expression pattern, we carried out unbiased microarray gene expression analysis and after induction of DNA damage and replication stress by radiation. ssDNA and dsDNA in the cytosolic fractions were quantified using Quant-iT OliGreen and PicoGreen Assay Kits. For cytoplasmic BrdU detection, exponentially grown cells were labeled with BrdU for 18-20 h and then immunostained with anti-BrdU antibody. Additionally, we measured the expression and post-translational modification of proteins involved in innate immune signaling by western blotting. We also employed DNA fiber assay to determine the role of Rad51 in replication fork processing. Results: We found that defects in Rad51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. Mechanistically, the unprotected newly replicated genome in the absence of Rad51 is degraded by the exonuclease activity of Mre11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data revealed that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, Rad51 is also implicated in the suppression of innate immunity. Conclusion: Rad51 plays a novel role in immunity outside its known functions in DSB repair and replication fork processing. We discovered that the lack of Rad51 leads to the upregulation of innate immune response pathway genes upon DNA damage and replication induced by irradiation. We found that in the absence of Rad51 the newly replicated genome is degraded by the exonuclease activity of Mre11. We also showed that these degraded nascent DNA fragments are exported to the cytoplasm, triggering innate immune response signaling. Our study reveals a previously unidentified role for Rad51 in triggering an innate immune response, and places Rad51 at the hub of new interconnections between DNA replication, DNA repair, and immunity. Funding: This work was supported by NIH R01AG053341 grants. Citation Format: Kalayarasan Srinivasan, Souparno Bhattacharya, Salim Abdisalaam, Shibani Mukherjee, Asaithamby Aroumougame. Rad51 suppresses innate immune response by blocking MRE11-mediated degradation of newly replicated genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2017-2490