Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging. Mutational characteristics of NER-deficiency may reveal important diagnostic opportunities, as tumors deficient in NER are more sensitive to certain treatments. Here, we analyzed the genome-wide somatic mutational profiles of adult stem cells (ASCs) from NER-deficient Ercc1 −/Δ mice. Our results indicate that NER-deficiency increases the base substitution load twofold in liver but not in small intestinal ASCs, which coincides with the tissue-specific aging pathology observed in these mice. Moreover, NER-deficient ASCs of both tissues show an increased contribution of Signature 8 mutations, which is a mutational pattern with unknown etiology that is recurrently observed in various cancer types. The scattered genomic distribution of the base substitutions indicates that deficiency of global-genome NER (GG-NER) underlies the observed mutational consequences. In line with this, we observe increased Signature 8 mutations in a GG-NER-deficient human organoid culture, in which XPC was deleted using CRISPR-Cas9 gene-editing. Furthermore, genomes of NER-deficient breast tumors show an increased contribution of Signature 8 mutations compared with NER-proficient tumors. Elevated levels of Signature 8 mutations could therefore contribute to a predictor of NER-deficiency based on a patient's mutational profile.
The small Tims chaperone hydrophobic precursors across the mitochondrial intermembrane space. Tim9 and Tim10 form the soluble TIM10 complex that binds precursors exiting from the outer membrane. Tim12 functions downstream, as the only small Tim peripherally attached on the inner membrane. We show that Tim12 has an intrinsic affinity for inner mitochondrial membrane lipids, in contrast to the other small Tims. We find that the C-terminal end of Tim12 is essential in vivo. Its deletion crucially abolishes assembly of Tim12 in complexes with the other Tims. The N-terminal end contains targeting information and also mediates direct binding of Tim12 to the transmembrane segments of the carrier substrates. These results provide a molecular basis for the concept that the essential role of Tim12 relies on its unique assembly properties that allow this subunit to bridge the soluble and membrane-embedded translocases in the carrier import pathway.The small Tims escort hydrophobic proteins of the mitochondrial inner membrane across the intermembrane space (IMS). 4 Key to their function is their organization in heterooligomeric complexes. Upon entering the outer membrane channel, the small Tims are recognized in the IMS by Mia40 (1). Site-specific oxidation of their cysteines into intramolecular disulfides releases them in an assembly-competent folded state that allows formation of specific Tim complexes (2-7).Tim12 stands out as the only small Tim exclusively located on the inner membrane (8, 9). It functions after recognition of the substrate by the soluble small Tim complexes (8, 9), and it associates with both the Tim9-Tim10 complex and Tim22 in the membrane (10). However, the molecular basis of its interactions is still unknown. Here, we show that Tim12 has an intrinsic affinity for inner mitochondrial membrane lipids, which are enriched in cardiolipin. Deletion analysis, in vivo complementation assays, and import assays followed by analysis of native Tim complexes revealed distinct functional domains in Tim12: the N-terminal segment contains necessary import information but is dispensable for function, whereas the C-terminal end is essential in vivo, has a determining role in lipid binding and association with Tim9, and affects the assembly of Tim12 in complexes with other Tims. We show that Tim12 directly binds to transmembrane segments of its substrates, primarily via its N-terminal domain. As this "substrate sensor" domain of Tim12 overlaps in substrate binding with that of Tim10 and is dispensable, whereas on the other hand the Tim12 C-terminal "assembly domain" is essential, we propose that the key feature of Tim12 function relies on its role as a linker subunit between the soluble and membrane-embedded translocase complexes. EXPERIMENTAL PROCEDURES Cloning of Tim12 VariantsIn Vitro Transcription/Translation pSP64 Vector-Wildtype TIM12 was PCR-amplified with Tim12 (forward BamHI) and Tim12 (reverse EcoRI) primers from a yeast genomic DNA and cloned into pSP64 with BamHI-EcoRI. The deletion mutants were then PCR-a...
Organs age differently, causing wide heterogeneity in multimorbidity, but underlying mechanisms are largely elusive. To investigate the basis of organ‐specific ageing, we utilized progeroid repair‐deficient Ercc1Δ/− mouse mutants and systematically compared at the tissue, stem cell and organoid level two organs representing ageing extremes. Ercc1Δ/− intestine shows hardly any accelerated ageing. Nevertheless, we found apoptosis and reduced numbers of intestinal stem cells (ISCs), but cell loss appears compensated by over‐proliferation. ISCs retain their organoid‐forming capacity, but organoids perform poorly in culture, compared with WT. Conversely, liver ages dramatically, even causing early death in Ercc1‐KO mice. Apoptosis, p21, polyploidization and proliferation of various (stem) cells were prominently elevated in Ercc1Δ/− liver and stem cell populations were either largely unaffected (Sox9+), or expanding (Lgr5+), but were functionally exhausted in organoid formation and development in vitro. Paradoxically, while intestine displays less ageing, repair in WT ISCs appears inferior to liver as shown by enhanced sensitivity to various DNA‐damaging agents, and lower lesion removal. Our findings reveal organ‐specific anti‐ageing strategies. Intestine, with short lifespan limiting time for damage accumulation and repair, favours apoptosis of damaged cells relying on ISC plasticity. Liver with low renewal rates depends more on repair pathways specifically protecting the transcribed compartment of the genome to promote sustained functionality and cell preservation. As shown before, the hematopoietic system with intermediate self‐renewal mainly invokes replication‐linked mechanisms, apoptosis and senescence. Hence, organs employ different genome maintenance strategies, explaining heterogeneity in organ ageing and the segmental nature of DNA‐repair‐deficient progerias.
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