Tissue-Specific Activation of Microhomology-Mediated End Joining with Age Reflects Dynamic Rewiring of DNA Repair in Rats

Genomic instability is a hallmark of ageing, driven by the accumulation of DNA lesions and the decline of high-fidelity repair pathways such as homologous recombination (HR) and classical nonhomologous end joining (c-NHEJ). We hypothesized that ageing cells increasingly rely on microhomology-mediated end joining (MMEJ), an inherently error-prone repair pathway, whose role in ageing remains poorly characterized. Here, we present the first systematic, tissue-wide analysis of MMEJ activity across eight rat organs using an in vitro assay with defined 10–16 nt microhomology substrates and cell-free extracts from rats of different ages. Our results reveal age-associated and tissue-specific reprogramming of MMEJ: activity increased in the testes and liver, newly emerged in kidneys and lungs, and declined in immune tissues such as the spleen and thymus. Strikingly, aged brain extracts exhibited latent MMEJ activity. Repair efficiency was further modulated by microhomology length in a tissue-dependent manner. These functional shifts correlated with altered expression of key MMEJ effectors, including XRCC1, PARP1, Ligase III, and notably FEN1, whose upregulation in aged lungs mirrored robust MMEJ activation. Together, our findings uncover dynamic remodelling of MMEJ during ageing, implicating it in age-related genomic instability and highlighting potential therapeutic targets to mitigate mutation burden in ageing tissues.