Stress-triggered hematopoietic stem cell proliferation relies on PrimPol-mediated repriming

p38 MAPK fuels proliferation stress and DNA damage impairing the functionality of genetically engineered hematopoietic stem and progenitor cells

“…4). Furthermore, we interrogated our RNA sequencing data to reveal possible changes in replication dynamics in the HSC/MPP cluster and found that p38 inhibition resulted in downregulation of the replication initiation and response to DNA damage and replication stress programs 22 , further supporting the findings that proliferation slowdown by p38i treatment mitigates culture-triggered DNA damage (Fig. 4g).…”

“…Even if we cannot completely rule out the involvement of p38MAPK in other aspects of culture adaptation, our study shows that longer G1 phase and G1/S transition are associated with reduced DNA damage and DDR levels after ex vivo culture, likely by conferring more time to repair DNA lesions before the following S-phase as suggested in murine embryonic stem cells, where delaying G1 progression suppresses ssDNA accumulation 24 . Moreover, in murine stem cells, it has been shown that uncontrolled proliferation driven by pI:pC in vivo administration (simulating a viral infection) modifies DNA replication patterns toward a PrimPol-mediated faster and discontinuous DNA synthesis, posing a risk to genome integrity and stability 22 . In our setting, reduced RPA and ATR phosphorylation, together with the downregulation of replication stress-related gene signatures, suggested that regulation of proliferation by prolonging cell cycle length might reduce DNA replication stress, likely limiting replication forks stalling.…”

“…We recently discovered that GE itself, coupled with ex vivo culture, culminates into a p53mediated DNA damage response (DDR) activation leading to cell differentiation, loss of engraftment, and reduced graft clonality 16,[19][20][21] . Importantly, a set of data mainly obtained in mouse settings indicates that proliferating stem cells accumulate elevated levels of DNA replication stress, manifesting in the accumulation of DNA single-strand breaks (SSBs) [22][23][24] , spontaneous mutations or chromosomal abnormalities, and production of reactive oxidative species (ROS) and metabolic changes [25][26][27] , ultimately resulting in stem cell dysfunction. Similarly, it was reported that other stimuli, such as osmotic stress and pro-inflammatory cytokines, may trigger the stress kinase p38 mitogen-activated protein kinase (MAPK), resulting in differentiation and loss of HSC selfrenewal 26,28,29 .…”

Polymerase iota (Pol ι) prevents PrimPol-mediated nascent DNA synthesis and chromosome instability

“…Several recent studies (43)(44)(45)(46)(47) reported a competition between PrimPol-mediated repriming and fork reversal. The depletion of the fork reversal promoting factor ZRANB3 (zinc finger RANBP2-type containing 3) (48) and of Pol ι have a similar and epistatic impact on the length of nascent DNA tracks (Fig.…”

Replication fork plasticity upon replication stress requires rapid nuclear actin polymerization

“…This discontinuous mode of replication promotes bypass of bulky DNA lesions and implies generation of ssDNA gaps that are filled post-replicatively to complete genome duplication [16][17][18][19][20] . Fork reversal and repriming are competing options of DNA damage tolerance 5 , and fine-tuning their balance recently proved crucial to determine the response to genotoxic treatments 10,21 , and to enable proliferation bursts upon tissue-specific stimuli 22 . Recent evidence suggested that fork plasticity transactions are not limited to forks directly challenged by DNA lesions or replication interference, but rather rapidly extend to unchallenged forks as a global, nuclear response 23 .…”