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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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of 'naked' or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8-24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization.

DNA double-strand breaks (DSBs) are extremely dangerous lesions with severe consequences for cell survival and the maintenance of genomic stability. In higher eukaryotic cells, DSBs in chromatin promptly initiate the phosphorylation of the histone H2A variant, H2AX, at Serine 139 to generate gamma-H2AX. This phosphorylation event requires the activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Regions in chromatin with gamma-H2AX are conveniently detected by immunofluorescence microscopy and serve as beacons of DSBs. This has allowed the development of an assay that has proved particularly useful in the molecular analysis of the processing of DSBs. Here, we first review the role of gamma-H2AX in DNA damage response in the context of chromatin and discuss subsequently the use of this modification as a surrogate marker for mechanistic studies of DSB induction and processing. We conclude with a critical analysis of the strengths and weaknesses of the approach and present some interesting applications of the resulting methodology.

The aim of the present in vitro study was to establish the minimum necessary curing time to bond stainless steel brackets (Mini Diamond Twin) using new, intensive, light-emitting diode (LED) curing units. Seventy-five bovine primary incisors were divided into five equal groups. A standard light curing adhesive (Transbond XT) was used to bond the stainless steel brackets using different lamps and curing times. Two groups were bonded using an intensive LED curing lamp (Ortholux LED) for 5 and 10 seconds. Two more groups were bonded using another intensive LED curing device (Ultra-Lume LED 5) also for 5 and 10 seconds. Finally, a high-output halogen lamp (Optilux 501) was used for 40 seconds to bond the final group, which served as a positive control. All teeth were fixed in hard acrylic and stored for 24 hours in water at 37 degrees C. Shear bond strength (SBS) was measured using an Instron testing machine. Weibull distribution and analysis of variance were used to test for significant differences. The SBS values obtained were significantly different between groups (P < 0.001). When used for 10 seconds, the intensive LED curing units achieved sufficient SBS, comparable with the control. In contrast, 5 seconds resulted in significantly lower SBS. The adhesive remnant index (ARI) was not significantly affected.A curing time of 10 seconds was found to be sufficient to bond metallic brackets to incisors using intensive LED curing units. These new, comparatively inexpensive, curing lamps seem to be an advantageous alternative to conventional halogen lamps for bonding orthodontic brackets.