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Charged-particle radiotherapy (CPRT) utilizing low and high linear energy transfer (low-/high-LET) ionizing radiation (IR) is a promising cancer treatment modality having unique physical energy deposition properties. CPRT enables focused delivery of a desired dose to the tumor, thus achieving a better tumor control and reduced normal tissue toxicity. It increases the overall radiation tolerance and the chances of survival for the patient. Further improvements in CPRT are expected from a better understanding of the mechanisms governing the biological effects of IR and their dependence on LET. There is increasing evidence that high-LET IR induces more complex and even clustered DNA double-strand breaks (DSBs) that are extremely consequential to cellular homeostasis, and which represent a considerable threat to genomic integrity. However, from the perspective of cancer management, the same DSB characteristics underpin the expected therapeutic benefit and are central to the rationale guiding current efforts for increased implementation of heavy ions (HI) in radiotherapy. Here, we review the specific cellular DNA damage responses (DDR) elicited by high-LET IR and compare them to those of low-LET IR. We emphasize differences in the forms of DSBs induced and their impact on DDR. Moreover, we analyze how the distinct initial forms of DSBs modulate the interplay between DSB repair pathways through the activation of DNA end resection. We postulate that at complex DSBs and DSB clusters, increased DNA end resection orchestrates an increased engagement of resection-dependent repair pathways. Furthermore, we summarize evidence that after exposure to high-LET IR, error-prone processes outcompete high fidelity homologous recombination (HR) through mechanisms that remain to be elucidated. Finally, we review the high-LET dependence of specific DDR-related post-translational modifications and the induction of apoptosis in cancer cells. We believe that in-depth characterization of the biological effects that are specific to high-LET IR will help to establish predictive and prognostic signatures for use in future individualized therapeutic strategies, and will enhance the prospects for the development of effective countermeasures for improved radiation protection during space travel.

Using data generated with cells exposed to ionizing-radiation (IR) in G 2 -phase of the cell cycle, we describe dose-dependent interactions between ATM, ATR and DNA-PKcs revealing unknown mechanistic underpinnings for two key facets of the DNA damage response: DSB end-resection and G 2 -checkpoint activation. At low IR-doses that induce low DSB-numbers in the genome, ATM and ATR regulate epistatically the G 2 -checkpoint, with ATR at the output-node, interfacing with the cell-cycle predominantly through Chk1. Strikingly, at low IR-doses, ATM and ATR epistatically regulate also resection, and inhibition of either activity fully suppresses resection. At high IR-doses that induce high DSB-numbers in the genome, the tight ATM/ATR coupling relaxes and independent outputs to G 2 -checkpoint and resection occur. Consequently, both kinases must be inhibited to fully suppress checkpoint activation and resection. DNA-PKcs integrates to the ATM/ATR module by regulating resection at all IR-doses, with defects in DNA-PKcs causing hyper-resection and G 2 -checkpoint hyper-activation. Notably, hyper-resection is absent from other c-NHEJ mutants. Thus, DNA-PKcs specifically regulates resection and adjusts the activation of the ATM/ATR module. We propose that selected DSBs are shepherd by DNA-PKcs from c-NHEJ to resection-dependent pathways for processing under the regulatory supervision of the ATM/ATR module.

DNA double-strand break (DSB) clusters are a hallmark of high-linear energy transfer (high-LET) radiation and are associated with pronounced biological effects, including reduced cell survival and elevated genomic instability. Our previous work in Chinese hamster cells, engineered with variably designed clusters of I-SceI recognition sites, integrated at multiple genomic locations, revealed that DSB clusters suppress classical non-homologous end-joining (c-NHEJ) and induce chromosomal translocations that ultimately increase cell lethality. Here, we extend this line of investigation to human cell lines and generate DSB clusters using alternative approaches that do not require prior genetic manipulation of the test cell lines. We employ CRISPR/Cas9-technology to generate DSB clusters of specific design at a selected genomic locus and examine their consequences on locus integrity. We target Exon 3 of the human HPRT ( hHPRT ) gene and introduce single DSBs or DSB clusters of varying numbers and inter-DSB distances. Alterations at the locus reflecting hHPRT gene inactivation, are quantified as mutations causing resistance to 6-thioguanine (6TG). Our results show that DSB clusters are markedly more potent inducers of mutations than single DSBs and that DSBs spaced within ~ 600 base pairs synergize in mutation induction. Mechanistic analyses using small-molecule inhibitors and engineered gene knockout cell lines reveal that the increased mutagenicity of clustered DSBs is primarily mediated by DNA end resection and PARP1-dependent alternative end-joining (alt-EJ) pathways. These findings reinforce the biological relevance of DSB clusters as a severe form of complex DNA damage and provide mechanistic insights into high-LET radiation-induced increased cell killing and genomic instability.

In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers.

The cell cycle-dependent engagement of DNA-end resection at DSBs is regulated by phosphorylation of CTIP by CDKs, the central regulators of cell cycle transitions. Cell cycle transitions are also intimately regulated by protein degradation via two E3 ubiquitin ligases: SCF SKP2 and APC/C CDH1 complex. Although APC/C CDH1 regulates CTIP in G 1 – and G 2 -phase, contributions by SCF SKP2 have not been reported. We demonstrate that SCF SKP2 is a strong positive regulator of resection. Knockdown of SKP2, fully suppresses resection in several cell lines. Notably, this suppression is G 2 -phase specific and is not observed in S-phase or G 1 –phase cells. Knockdown of SKP2 inactivates SCF SKP2 causing APC/C CDH1 activation, which degrades CTIP. The stabilizing function of SCF SKP2 on CTIP promotes resection and supports gene conversion (GC), alternative end joining (alt-EJ) and cell survival. We propose that CDKs and SCF SKP2 -APC/C CDH1 cooperate to regulate resection and repair pathway choice at DSBs in G 2 -phase.

Heavy-ion radiotherapy utilizing high linear energy transfer (high-LET) ionizing radiation (IR) is a promising cancer treatment modality owing to advantageous physical properties of energy deposition and associated toxicity over X-rays. Therapies utilizing high-LET radiation will benefit from a better understanding of the molecular mechanisms underpinning their increased biological efficacy. Towards this goal, we investigate here the biological consequences of well-defined clusters of DNA double-strand breaks (DSBs), a form of DNA damage, which on theoretical counts, has often been considered central to the enhanced toxicity of high-LET IR. We test clonal cell lines harboring in their genomes constructs with appropriately engineered I-SceI recognition sites that convert upon I- Sce I expression to individual DSBs, or DSB-clusters comprising known numbers of DSBs with defined DNA-ends. We find that, similarly to high-LET IR, DSB-clusters of increasing complexity, i.e. increasing numbers of DSBs, with compatible or incompatible ends, compromise classical non-homologous end-joining, favor DNA end-resection and promote resection-dependent DSB-processing. Analysis of RAD51 foci shows increased engagement of error-free homologous recombination on DSB-clusters. Multicolor fluorescence in situ hybridization analysis shows that complex DSB-clusters markedly increase the incidence of structural chromosomal abnormalities (SCAs). Since RAD51-knockdown further increases SCAs-incidence, we conclude that homologous recombination suppresses SCAs-formation. Strikingly, CtIP-depletion inhibits SCAs-formation, suggesting that it relies on alternative end-joining or single-strand annealing. Indeed, ablation of RAD52 causes a marked reduction in SCAs, as does also inhibition of PARP1. We conclude that increased DSB-cluster formation that accompanies LET-increases, enhances IR-effectiveness by promoting DNA end-resection, which suppresses c-NHEJ and enhances utilization of alt-EJ or SSA. Although increased resection also favors HR, on balance, error-prone processing dominates, causing the generally observed increased toxicity of high-LET radiation. These findings offer new mechanistic insights into high-LET IR-toxicity and have translational potential in the clinical setting that may be harnessed by combining high-LET IR with inhibitors of PARP1 or RAD52.

The utilization of high linear-energy-transfer (LET) ionizing radiation (IR) modalities is rapidly growing worldwide, causing excitement but also raising concerns, because our understanding of their biological effects is incomplete. Charged particles such as protons and heavy ions have increasing potential in cancer therapy, due to their advantageous physical properties over X-rays (photons), but are also present in the space environment, adding to the health risks of space missions. Therapy improvements and the protection of humans during space travel will benefit from a better understanding of the mechanisms underpinning the biological effects of high-LET IR. There is evidence that high-LET IR induces DNA double-strand breaks (DSBs) of increasing complexity, causing enhanced cell killing, owing, at least partly, to the frequent engagement of a low-fidelity DSB-repair pathway: alternative end-joining (alt-EJ), which is known to frequently induce severe structural chromosomal abnormalities (SCAs). Here, we evaluate the radiosensitivity of A549 lung adenocarcinoma cells to X-rays, α-particles and 56Fe ions, as well as of HCT116 colorectal cancer cells to X-rays and α-particles. We observe the expected increase in cell killing following high-LET irradiation that correlates with the increased formation of SCAs as detected by mFISH. Furthermore, we report that cells exposed to low doses of α-particles and 56Fe ions show an enhanced G2-checkpoint response which is mainly regulated by ATR, rather than the coordinated ATM/ATR-dependent regulation observed after exposure to low doses of X-rays. These observations advance our understanding of the mechanisms underpinning high-LET IR effects, and suggest the potential utility for ATR inhibitors in high-LET radiation therapy.

Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs) from their genomes predominantly by two non-homologous end joining (NHEJ) pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK) and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of the alternative, backup NHEJ are less well characterized. Notably, the contribution of LIG1 to alternative NHEJ remains conjectural and although biochemical, cytogenetic and genetic experiments implicate LIG3, this contribution has not been formally demonstrated. Here, we take advantage of the powerful genetics of the DT40 chicken B-cell system to delineate the roles of LIG1 and LIG3 in alternative NHEJ. Our results expand the functions of LIG1 to alternative NHEJ and demonstrate a remarkable ability for LIG3 to backup DSB repair by NHEJ in addition to its essential function in the mitochondria. Together with results on DNA replication, these observations uncover a remarkable and previously unappreciated functional flexibility and interchangeability between LIG1 and LIG3.

We have recently reported that in G2-phase cells (but not S-phase cells) sustaining low loads of DNA double-strand break (DSBs), ATM and ATR regulate the G2-checkpoint epistatically, with ATR at the output-node, interfacing with the cell cycle through Chk1. However, although inhibition of ATR nearly completely abrogated the checkpoint, inhibition of Chk1 using UCN-01 generated only partial responses. This suggested that additional kinases downstream of ATR were involved in the transmission of the signal to the cell cycle engine. Additionally, the broad spectrum of kinases inhibited by UCN-01 pointed to uncertainties in the interpretation that warranted further investigations. Here, we show that more specific Chk1 inhibitors exert an even weaker effect on G2-checkpoint, as compared to ATR inhibitors and UCN-01, and identify the MAPK p38α and its downstream target MK2 as checkpoint effectors operating as backup to Chk1. These observations further expand the spectrum of p38/MK2 signaling to G2-checkpoint activation, extend similar studies in cells exposed to other DNA damaging agents and consolidate a role of p38/MK2 as a backup kinase module, adding to similar backup functions exerted in p53 deficient cells. The results extend the spectrum of actionable strategies and targets in current efforts to enhance the radiosensitivity in tumor cells.

Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to benefit from DNA end-resection—a process whereby 3′ single-stranded DNA-tails are generated—initiated by the CtIP/MRE11-RAD50-NBS1 (MRN) complex and extended by EXO1 or the BLM/DNA2 complex. The connection between alt-EJ and resection remains incompletely characterized. Alt-EJ depends on the cell cycle phase, is at maximum in G2-phase, substantially reduced in G1-phase and almost undetectable in quiescent, G0-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G1- and G0-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G1-phase cells allow modest resection and alt-EJ, as compared to G2-phase cells. Strikingly, CtIP is undetectable in G0-phase cells owing to APC/C-mediated degradation. The suppression of CtIP degradation with bortezomib or CDH1-depletion rescues CtIP and alt-EJ in G0-phase cells. CtIP activation in G0-phase cells also requires CDK-dependent phosphorylation by any available CDK but is restricted to CDK4/6 at the early stages of the normal cell cycle. We suggest that suppression of mutagenic alt-EJ in G0-phase is a mechanism by which cells of higher eukaryotes maintain genomic stability in a large fraction of non-cycling cells in their organisms.