Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks.

We recently reported that when low doses of ionizing radiation induce low numbers of DNA double-strand breaks (DSBs) in G2-phase cells, about 50 % of them are repaired by homologous recombination (HR) and the remaining by classical non-homologous end-joining (c-NHEJ). However, with increasing DSB-load, the contribution of HR drops to undetectable (at ∼10 Gy) as c-NHEJ dominates. It remains unknown whether the approximately equal shunting of DSBs between HR and c-NHEJ at low radiation doses and the predominant shunting to c-NHEJ at high doses, applies to every DSB, or whether the individual characteristics of each DSB generate processing preferences. When G2-phase cells are irradiated, only about 10 % of the induced DSBs break the chromatids. This breakage allows analysis of the processing of this specific subset of DSBs using cytogenetic methods. Notably, at low radiation doses, these DSBs are almost exclusively processed by HR, suggesting that chromatin characteristics awaiting characterization underpin chromatid breakage and determine the preferential engagement of HR. Strikingly, we also discovered that with increasing radiation dose, a pathway switch to c-NHEJ occurs in the processing of this subset of DSBs. Here, we confirm and substantially extend our initial observations using additional methodologies. Wild-type cells, as well as HR and c-NHEJ mutants, are exposed to a broad spectrum of radiation doses and their response analyzed specifically in G2 phase. Our results further consolidate the observation that at doses <2 Gy, HR is the main option in the processing of the subset of DSBs generating chromatid breaks and that a pathway switch at doses between 4-6 Gy allows the progressive engagement of c-NHEJ. PARP1 inhibition, irrespective of radiation dose, leaves chromatid break repair unaffected suggesting that the contribution of alternative end-joining is undetectable under these experimental conditions.

Chromosome breaks generated by low doses of ionizing radiation in G2-phase are processed exclusively by gene conversion.

Four repair pathways process DNA double-strand breaks (DSBs). Among these pathways the homologous recombination repair (HRR) subpathway of gene conversion (GC) affords error-free processing, but functions only in S- and G2-phases of the cell cycle. Classical non-homologous end-joining (c-NHEJ) operates throughout the cell cycle, but causes small deletions and translocations. Similar deficiencies in exaggerated form, combined with reduced efficiency, are associated with alternative end-joining (alt-EJ). Finally, single-strand annealing (SSA) causes large deletions and possibly translocations. Thus, processing of a DSB by any pathway, except GC, poses significant risks to the genome, making the mechanisms navigating pathway-engagement critical to genome stability. Logically, the cell ought to attempt engagement of the pathway ensuring preservation of the genome, while accommodating necessities generated by the types of DSBs induced. Thereby, inception of DNA end-resection will be key determinant for GC, SSA and alt-EJ engagement. We reported that during G2-phase, where all pathways are active, GC engages in the processing of almost 50 % of DSBs, at low DSB-loads in the genome, and that this contribution rapidly drops to nearly zero with increasing DSB-loads. At the transition between these two extremes, SSA and alt-EJ compensate, but at extremely high DSB-loads resection-dependent pathways are suppressed and c-NHEJ remains mainly active. We inquired whether in this processing framework all DSBs have similar fates. Here, we analyze in G2-phase the processing of a subset of DSBs defined by their ability to break chromosomes. Our results reveal an absolute requirement for GC in the processing of chromatid breaks at doses in the range of 1 Gy. Defects in c-NHEJ delay significantly the inception of processing by GC, but leave processing kinetics unchanged. These results delineate the essential role of GC in chromatid break repair before mitosis and classify DSBs that underpin this breakage as the exclusive substrate of GC.

Strong suppression of gene conversion with increasing DNA double-strand break load delimited by 53BP1 and RAD52.

In vertebrates, genomic DNA double-strand breaks (DSBs) are removed by non-homologous end-joining processes: classical non-homologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ); or by homology-dependent processes: gene-conversion (GC) and single-strand annealing (SSA). Surprisingly, these repair pathways are not real alternative options restoring genome integrity with equal efficiency, but show instead striking differences in speed, accuracy and cell-cycle-phase dependence. As a consequence, engagement of one pathway may be associated with processing-risks for the genome absent from another pathway. Characterization of engagement-parameters and their consequences is, therefore, essential for understanding effects on the genome of DSB-inducing agents, such as ionizing-radiation (IR). Here, by addressing pathway selection in G2-phase, we discover regulatory confinements in GC with consequences for SSA- and c-NHEJ-engagement. We show pronounced suppression of GC with increasing DSB-load that is not due to RAD51 availability and which is delimited but not defined by 53BP1 and RAD52. Strikingly, at low DSB-loads, GC repairs ∼50% of DSBs, whereas at high DSB-loads its contribution is undetectable. Notably, with increasing DSB-load and the associated suppression of GC, SSA gains ground, while alt-EJ is suppressed. These observations explain earlier, apparently contradictory results and advance our understanding of logic and mechanisms underpinning the wiring between DSB repair pathways.

A method for the cell-cycle-specific analysis of radiation-induced chromosome aberrations and breaks.

The classical G2-assay is widely used to assess cell-radiosensitivity and cancer phenotype: Cells are exposed to low doses of ionizing-radiation (IR) and collected for cytogenetic- analysis ˜1.5 h later. In this way, chromosome-damage is measured in cells irradiated in G2-phase, without retrieving information regarding kinetics of chromosome-break-repair. Modification of the assay to include analysis at multiple time-points after IR, has enabled kinetic-analysis of chromatid-break-repair and assessment of damage in a larger proportion of G2-phase cells. This modification, however, increases the probability that at later time points not only cells irradiated in G2-phase, but also cells irradiated in S-phase will reach metaphase. However, the response of cells irradiated in G2-phase can be mechanistically different from that of cells irradiated in S-phase. Therefore, indiscriminate analysis may confound the interpretation of experiments designed to elucidate mechanisms of chromosome-break-repair and the contributions of the different DSB-repair-pathways in this response. Here we report an EdU based modification of the assay that enables S- and G2-phase specific analysis of chromatid break repair. Our results show that the majority of metaphases captured during the first 2 h after IR originate from cells irradiated in G2-phase (EdU- metaphases) in both rodent and human cells. Metaphases originating from cells irradiated in S-phase (EdU+ metaphases) start appearing at 2 h and 4 h after IR in rodent and human cells, respectively. The kinetics of chromatid-break-repair are similar in cells irradiated in G2- and S-phase of the cell-cycle, both in rodent and human cells. The protocol is applicable to classical-cytogenetic experiments and allows the cell-cycle specific analysis of chromosomal-aberrations. Finally, the protocol can be applied to the kinetic analysis of chromosome-breaks in prematurely-condensed-chromosomes of G2-phase cells. In summary, the developed protocol provides means to enhance the analysis of IR-induced-cytogenetic-damage by providing information on the cell-cycle phase where DNA damage is inflicted.