DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonucleasedeficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linkerinduced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.T he tumor suppressor protein p53 has been called the guardianof-the-genome due to its ability to transactivate downstream targets transcriptionally, which prevents S-phase entrance before facilitating DNA repair or eliminating cells with severe DNA damage via apoptosis (1). Interestingly, p53 also encodes an intrinsic 3′-5′ exonuclease activity located within its central DNA-binding domain (2-4). The contribution of the exonuclease proficiency to p53's function has largely remained obscure. Exonucleases are involved in DNA replication, DNA repair, and recombination, increasing the fidelity or efficiency of these processes. The 3′-5′ exonuclease activity of DNA polymerases (POLs) catalyzes the correction of replication errors, thereby preventing genomic instability and cancer (5-7). The potential involvement of p53's exonuclease in DNA repair has been ascribed to transcription-independent functions in nucleotide excision repair and base excision repair, in homologous recombination (HR), and in mitochondrial processes (8-10).Regarding HR, in particular, reports indicate a dual role for p53. On the one hand, it has been reported that p53 down-regulates unscheduled and excessive HR in response to severe genotoxic stress, like formation of DNA double-strand breaks (DSBs) (8-10). This antirecombinogenic effect of p53 has been linked to the blockage of continued strand exchange by interactions with recombinase RAD51, RAD54, and nascent HR intermediates carrying specific mismatches (11, 12). On the other hand, p53 stimulates spontaneous HR during S-phase to overcome replication fork stalling and to pr...

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The breakpoint cluster region of the MLL gene (MLLbcr) is frequently rearranged in therapy-related and infant acute leukaemia, but the destabilizing mechanism is poorly understood. We recently proposed that DNA replication stress results in MLLbcr cleavage via endonuclease G (EndoG) and represents the common denominator of genotoxic therapy-induced MLL destabilization. Here we performed a siRNA screen for new factors involved in replication stress-induced MLL rearrangements employing an enhanced green fluorescent protein-based reporter system. We identified 10 factors acting in line with EndoG in MLLbcr breakage or further downstream in the repair of the MLLbcr breaks, including activation-induced cytidine deaminase (AID), previously proposed to initiate MLLbcr rearrangements in an RNA transcription-dependent mechanism. Further analysis connected AID and EndoG in MLLbcr destabilization via base excision repair (BER) components. We show that replication stress-induced recruitment of EndoG to the MLLbcr and cleavage are AID/BER dependent. Notably, inhibition of the core BER factor Apurinic-apyrimidinic endonuclease 1 protects against MLLbcr cleavage in tumour and human cord blood-derived haematopoietic stem/progenitor cells, harbouring the cells of origin of leukaemia. We propose that off-target binding of AID to the MLLbcr initiates BER-mediated single-stranded DNA cleavage, which causes derailed EndoG activity ultimately resulting in leukaemogenic MLLbcr rearrangements.

Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment.

Quercetin (Que) is an abundant flavonoid in the human diet and high-concentration food supplement with reported pro- and anti-carcinogenic activities. Topoisomerase II (TopoII) inhibition and subsequent DNA damage induction by Que was implicated in the mixed lineage leukemia gene (MLL) rearrangements that can induce infant and adult leukemias. This notion raised concerns regarding possible genotoxicities of Que in hematopoietic stem and progenitor cells (HSPCs). However, molecular targets mediating Que effects on DNA repair relevant to MLL translocations have not been defined. In this study we describe novel and potentially genotoxic Que activities in suppressing non-homologous end joining and homologous recombination pathways downstream of MLL cleavage. Using pharmacological dissection of DNA-PK, ATM and PI3K signalling we defined PI3K inhibition by Que with a concomitant decrease in the abundance of key DNA repair genes to be responsible for DNA repair inhibition. Evidence for the downstream TopoII-independent mutagenic potential of Que was obtained by documenting further increased frequencies of MLL rearrangements in human HSPCs concomitantly treated with Etoposide and Que versus single treatments. Importantly, by engaging a tissue engineered placental barrier, we have established the extent of Que transplacental transfer and hence provided the evidence for Que reaching fetal HSPCs. Thus, Que exhibits genotoxic effects in human HSPCs via different mechanisms when applied continuously and at high concentrations. In light of the demonstrated Que transfer to the fetal compartment our findings are key to understanding the mechanisms underlying infant leukemia and provide molecular markers for the development of safety values.

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 ATMdependent 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. Lifelong blood production depends on HSPCs-a subset of primitive hematopoietic cells endowed with high self-renewal potential. HSPCs give rise to CPs with limited or no self-renewal, which in turn, differentiate into various mature blood cells. Analysis of human HSPC isolated from newborn, young, and elderly individuals by DNA sequencing has revealed that HSPCs serve as a reservoir for genetic changes, including mutations in genes implicated in leukemia; thus, they are a likely cell of origin for hematopoietic malignancies 1-5. DNA replication and cellular metabolism are among the endogenous sources of DNA damage that can contribute to mutagenesis and carcinogenesis. However, exposing the body to exogenous inducers of DNA damage, such as IR and certain chemotherapeutic drugs can greatly increase the rate and occurrence of genomic aberrations. Thus, these inducers are implicated in the development of bone marrow failure, myelodysplastic syndrome as well as de novo and therapy-related leukemia 6,7. DNA Double Strand Breaks (DSBs) are the most lethal and dangerous forms of DNA damage induced by IR, and when left unrepaired or misrepaired, they can lead to cell death or potentially

DNA double strand breaks (DSBs) are the most dangerous genomic lesions that can be induced by endogenous and exogenous sources. DNA damage response determines cellular fate decisions following DSBs and can lead to cell death or cell survival. Incorrect DSB repair via canonical Non-Homologous End Joining (cNHEJ) or Alternative NHEJ (Alt-NHEJ) is the main source of oncogenic aberrations, including leukemogenic translocations, DNA sequence deletions and insertions. The long life span of Hematopoietic Stem Cells (HSC) and their practically unlimited potential for self-renewal requires efficient strategies to cope with DNA damage to eliminate erroneous genetic information inheritance to daughter cells. Although the critical importance of maintaining genome integrity for normal hematopoiesis and prevention of leukemogenesis has been established, definitive analysis of DNA damage response and its mutagenic outcomes in human HSC and Progenitors in response to DSBs is missing. Here we repot that human cord blood purified HSC (defined as CD34+CD38-CD45RA-) are exquisitely sensitive to irradiation (IR)-induced apoptosis in contrast to committed progenitors (defined as CD34+CD38+) as validated by PARP cleavage induction. Interestingly, pan-caspase inhibitor Z-VAD-FMK prevented, whereas CHK2 inhibitor (PV1019) failed in altering apoptosis onset of irradiated HSC. Strikingly, CHK2 inhibitor blocked IR-induced apoptosis in cycling HSC, suggesting differential wiring of DNA damage induced apoptosis in quiescent versus mitogenically stimulated HSC. To characterize cNHEJ repair pathway and its mutagenic potential in live primitive hematopoietic cells we analyzed I-SceI endonuclease induced tandem DSBs joining capacity using DNA repair reporter assay. We found that HSC exhibit inferior cNHEJ capacity as compared with committed progenitors. By decreasing DSBs persistence we revealed that progenitors utilize to the higher degree than HSC the mutagenic component of cNHEJ pathway that results in DNA deletions. We identified HSC-specific contribution of CHK2 kinase activity in limiting incorrect DNA ends joining. Blockade of apoptosis induction also led to the selective increase in mutagenic NHEJ in HSC. On the other hand, inhibition of DNA-PK led to increased oncogenic repair in progenitors only. Importantly, we revealed that HSC utilized mutagenic Alt-NHEJ pathway that depends on microhomologies search and extensive DNA ends processing less efficiently than Progenitors. Thus, our results indicate that oncogenic consequences of DSBs repair in HSC are distinctly minimized by the non-redundant cell death and CHK2 dependent mechanisms. More broadly, these findings will help to elucidate additional repair modifiers and the mechanism by which HSC contend with genotoxic stress. Disclosures No relevant conflicts of interest to declare.

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