Replication stress is a potent driver of functional decline in ageing haematopoietic stem cells.

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age. Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation. While many drivers of HSC ageing have been proposed, the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated γH2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar γH2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.

Calcium-binding capacity of centrin2 is required for linear POC5 assembly but not for nucleotide excision repair.

Centrosomes, the principal microtubule-organising centres in animal cells, contain centrins, small, conserved calcium-binding proteins unique to eukaryotes. Centrin2 binds to xeroderma pigmentosum group C protein (XPC), stabilising it, and its presence slightly increases nucleotide excision repair (NER) activity in vitro. In previous work, we deleted all three centrin isoforms present in chicken DT40 cells and observed delayed repair of UV-induced DNA lesions, but no centrosome abnormalities. Here, we explore how centrin2 controls NER. In the centrin null cells, we expressed centrin2 mutants that cannot bind calcium or that lack sites for phosphorylation by regulatory kinases. Expression of any of these mutants restored the UV sensitivity of centrin null cells to normal as effectively as expression of wild-type centrin. However, calcium-binding-deficient and T118A mutants showed greatly compromised localisation to centrosomes. XPC recruitment to laser-induced UV-like lesions was only slightly slower in centrin-deficient cells than in controls, and levels of XPC and its partner HRAD23B were unaffected by centrin deficiency. Interestingly, we found that overexpression of the centrin interactor POC5 leads to the assembly of linear, centrin-dependent structures that recruit other centrosomal proteins such as PCM-1 and NEDD1. Together, these observations suggest that assembly of centrins into complex structures requires calcium binding capacity, but that such assembly is not required for centrin activity in NER.

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.

Centriole separation in DNA damage-induced centrosome amplification.

Altered centrosome numbers are seen in tumor cells in response to DNA damaging treatments and are hypothesised to contribute to cancer development. The mechanism by which the centrosome and chromosome cycles become disconnected after DNA damage is not yet clear. Here, we show that centrosome amplification occurs after ionising radiation (IR) in chicken DT40 cells that lack DNA-PK, Ku70, H2AX, Xpa, and Scc1, demonstrating that these activities are not required for centrosome amplification. We show that inhibition of topoisomerase II induces Chk1-dependent centrosome amplification, a similar response to that seen after IR. In the immortalised, nontransformed hTERT-RPE1 line, we observed centriole splitting, followed by dose-dependent centrosome amplification, after IR. We found that IR results in the formation of single, not multiple, daughter centrioles during centrosome amplification in U2OS osteosarcoma cells. Analysis of BRCA1 and BRCA2 mutant tumor cells showed high levels of centriole splitting in the absence of any treatment. IR caused pronounced levels of centrosome amplification in BRCA1 mutant breast cancer cells. These data show that centrosome amplification occurs after different forms of DNA damage in chicken cells, in nontransformed human cells and in human tumor cell lines, indicating that this is a general response to DNA damaging treatments. Together, our data suggest that centriole splitting is a key step in potentiation of the centrosome amplification that is a general response to DNA damage.