DEFINED BIOLOGICAL MODELS OF HIGH-LET RADIATION LESIONS.

DNA double-strand break (DSB) complexity is invoked to explain the increased efficacy of high-linear energy transfer (LET) radiation. Complexity is usually defined as presence of additional lesions in the immediate proximity of the DSB. DSB-clusters represent a different level of complexity that can jeopardize processing by destabilizing chromatin in the vicinity of the cluster. DSB-clusters are generated after exposure of cells to ionizing radiation (IR), particularly high-LET radiation, and have been considered as particularly consequential in several mathematical models of IR action. Yet, experimental demonstration of their relevance to the adverse IR effects, as well as information on the mechanisms underpinning their severity as DNA lesions is lacking. We addressed this void by developing cell lines with especially designed, multiply integrated constructs modeling defined combinations of DSB-clusters through appropriately engineered I-SceI meganuclease recognition sites. Using this model system, we demonstrate efficient activation of the DNA damage response, as well as a markedly increased potential of DSB-clusters, as compared to single-DSBs, to kill cells, and cause Parp1- dependent chromosomal translocations. We propose that DSB repair relying on first line DSB-processing pathways (canonical non-homologous end joining and to some degree homologous recombination repair) is compromised within DSB clusters, presumably through the associated chromatin destabilization, leaving alternative end joining as last option and translocation formation as a natural consequence. Our observations offer a mechanistic explanation for the increased efficacy of high-LET radiation.

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice.

Although the DNA double-strand break (DSB) is defined as a rupture in the double-stranded DNA molecule that can occur without chemical modification in any of the constituent building blocks, it is recognized that this form is restricted to enzyme-induced DSBs. DSBs generated by physical or chemical agents can include at the break site a spectrum of base alterations (lesions). The nature and number of such chemical alterations define the complexity of the DSB and are considered putative determinants for repair pathway choice and the probability that errors will occur during this processing. As the pathways engaged in DSB processing show distinct and frequently inherent propensities for errors, pathway choice also defines the error-levels cells opt to accept. Here, we present a classification of DSBs on the basis of increasing complexity and discuss how complexity may affect processing, as well as how it may cause lethal or carcinogenic processing errors. By critically analyzing the characteristics of DSB repair pathways, we suggest that all repair pathways can in principle remove lesions clustering at the DSB but are likely to fail when they encounter clusters of DSBs that cause a local form of chromothripsis. In the same framework, we also analyze the rational of DSB repair pathway choice.