Failure to precisely repair DNA damage in self-renewing Hematopoietic Stem and early Progenitor Cells (HSPCs) can disrupt normal hematopoiesis and promote leukemogenesis. Although HSPCs are widely considered a target of ionizing radiation (IR)-induced hematopoietic injury, definitive data regarding cell death, DNA repair, and genomic stability in these rare quiescent cells are scarce. We found that irradiated HSPCs, but not lineage-committed progenitors (CPs), undergo rapid ATM-dependent apoptosis, which is suppressed upon interaction with bone-marrow stroma cells. Using DNA repair reporters to quantify mutagenic Non-Homologous End Joining (NHEJ) processes, we found that HSPCs exhibit reduced NHEJ activities in comparison with CPs. HSPC-stroma interactions did not affect the NHEJ capacity of HSPCs, emphasizing its cell autonomous regulation. We noted diminished expression of multiple double strand break (DSB) repair transcripts along with more persistent 53BP1 foci in irradiated HSPCs in comparison with CPs, which can account for low NHEJ activity and its distinct control in HSPCs. Finally, we documented clonal chromosomal aberrations in 10% of IR-surviving HSPCs. Taken together, our results revealed potential mechanisms contributing to the inherent susceptibility of human HSPC to the cytotoxic and mutagenic effects of DNA damage.

The gender gap in life expectancy and cancer incidence suggests differences in the aging process between the sexes. Genomic instability has been recognized as a key factor in aging, but little is known about sex-specific differences. Therefore, we analyzed DNA double-strand break (DSB) repair in cycling human peripheral blood lymphocytes (PBL) from male and female donors of different age. Reporter-based DSB repair analyses revealed differential regulation of pathway usage in PBL from male and female donors with age: Non-homologous end joining (NHEJ) was inversely regulated in men and women; the activity of pathways requiring end processing and strand annealing steps such as microhomology-mediated end joining (MMEJ) declined with age in women but not in men. Screening candidate proteins identified the NHEJ protein KU70 as well as the end resection regulatory factors ATM and BLM showing reduced expression during aging in women. Consistently, the regulatory factor BLM contributed to the MMEJ proficiency in young but not in old women as demonstrated by knockdown analysis. In conclusion, we show that DSB repair is subject to changes upon aging and age-related changes in DSB repair are distinct in men and women.

The recently discovered p53-dependent DNA damage tolerance (DDT) pathway relies on its biochemical activities in DNA-binding, oligomerization, as well as complex formation with the translesion synthesis (TLS) polymerase iota (POLι). These p53-POLι complexes slow down nascent DNA synthesis for safe, homology-directed bypass of DNA replication barriers. In this study, we demonstrate that the alternative p53-isoforms p53β, p53γ, Δ40p53α, Δ133p53α, and Δ160p53α differentially affect this p53-POLι-dependent DDT pathway originally described for canonical p53α. We show that the C-terminal isoforms p53β and p53γ, comprising a truncated oligomerization domain (OD), bind PCNA. Conversely, N-terminally truncated isoforms have a reduced capacity to engage in this interaction. Regardless of the specific loss of biochemical activities required for this DDT pathway, all alternative isoforms were impaired in promoting POLι recruitment to PCNA in the chromatin and in decelerating DNA replication under conditions of enforced replication stress after Mitomycin C (MMC) treatment. Consistent with this, all alternative p53-isoforms no longer stimulated recombination, i.e., bypass of endogenous replication barriers. Different from the other isoforms, Δ133p53α and Δ160p53α caused a severe DNA replication problem, namely fork stalling even in untreated cells. Co-expression of each alternative p53-isoform together with p53α exacerbated the DDT pathway defects, unveiling impaired POLι recruitment and replication deceleration already under unperturbed conditions. Such an inhibitory effect on p53α was particularly pronounced in cells co-expressing Δ133p53α or Δ160p53α. Notably, this effect became evident after the expression of the isoforms in tumor cells, as well as after the knockdown of endogenous isoforms in human hematopoietic stem and progenitor cells. In summary, mimicking the situation found to be associated with many cancer types and stem cells, i.e., co-expression of alternative p53-isoforms with p53α, carved out interference with p53α functions in the p53-POLι-dependent DDT pathway.

The DNA damage response involves a complex protein network with members mediating different post-translational modifications such as ubiquitination and deubiquitination. Thereby the E3 ubiquitin ligase DTX3L as well as the deubiquitinase USP28 are recruited especially to DNA double strand breaks (DSBs) suggesting mutual functional interactions. Here we present evidence for the existence of such crosstalk. Mechanistically we show that DTX3L interacts directly with USP28 and ubiquitinates it, which leads to its proteasomal degradation. Vice versa, USP28 can remove those polyubiquitin chains from itself as well as from autoubiquitinated DTX3L. Consequently, these mutual regulatory interactions between DTX3L and USP28 affected DSB repair activities. Analysis of distinct DSB repair pathways reveals synthetic dysfunction of canonical non-homologues end joining (NHEJ) and homologous recombination (HR), upon USP28 and DTX3L double knockdown, suggesting cooperation between these proteins. Conversely, error-prone microhomology-mediated end joining (MMEJ) requires USP28 to counterbalance the antagonistic DTX3L effect. Together, the present data indicate that DTX3L and USP28 are under mutual control to fine-tune the capacity and quality of the cellular responses to stresses such as DNA damage.

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