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.

Generating realistic scaled complex networks.

Research on generative models plays a central role in the emerging field of network science, studying how statistical patterns found in real networks could be generated by formal rules. Output from these generative models is then the basis for designing and evaluating computational methods on networks including verification and simulation studies. During the last two decades, a variety of models has been proposed with an ultimate goal of achieving comprehensive realism for the generated networks. In this study, we (a) introduce a new generator, termed ReCoN; (b) explore how ReCoN and some existing models can be fitted to an original network to produce a structurally similar replica, (c) use ReCoN to produce networks much larger than the original exemplar, and finally (d) discuss open problems and promising research directions. In a comparative experimental study, we find that ReCoN is often superior to many other state-of-the-art network generation methods. We argue that ReCoN is a scalable and effective tool for modeling a given network while preserving important properties at both micro- and macroscopic scales, and for scaling the exemplar data by orders of magnitude in size.

Inhibition of Homologous Recombination and Promotion of Mutagenic Repair of DNA Double-Strand Breaks Underpins Arabinoside-Nucleoside Analogue Radiosensitization.

In concurrent chemoradiotherapy, drugs are used to sensitize tumors to ionizing radiation. Although a spectrum of indications for simultaneous treatment with drugs and radiation has been defined, the molecular mechanisms underpinning tumor radiosensitization remain incompletely characterized for several such combinations. Here, we investigate the mechanisms of radiosensitization by the arabinoside nucleoside analogue 9-ß-D-arabinofuranosyladenine (araA) placing particular emphasis on the repair of DNA double-strand breaks (DSB), and compare the results to those obtained with fludarabine (F-araA) and cytarabine (araC). Postirradiation treatment with araA strongly sensitizes cells to ionizing radiation, but leaves unchanged DSB repair by NHEJ in logarithmically growing cells, in sorted G1 or G2 phase populations, as well as in cells in the plateau phase of growth. Notably, araA strongly inhibits DSB repair by homologous recombination (HRR), as assessed by scoring ionizing radiation-induced RAD51 foci, and in functional assays using integrated reporter constructs. Cells compromised in HRR by RNAi-mediated transient knockdown of RAD51 show markedly reduced radiosensitization after treatment with araA. Remarkably, mutagenic DSB repair compensates for HRR inhibition in araA-treated cells. Compared with araA, F-araA and araC are only modestly radiosensitizing under the conditions examined. We propose that the radiosensitizing potential of nucleoside analogues is linked to their ability to inhibit HRR and concomitantly promote the error-prone processing of DSBs. Our observations pave the way to treatment strategies harnessing the selective inhibitory potential of nucleoside analogues and the development of novel compounds specifically utilizing HRR inhibition as a means of tumor cell radiosensitization.

Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair.

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.

Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.

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.