Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks

In mammalian cells, DNA double-strand breaks (DSBs) are repaired by three pathways, nonhomologous end-joining (NHEJ), gene conversion (GC) and single-strand annealing (SSA). These pathways are distinct with regard to repair efficiency and mutagenic potential and must be tightly controlled to preserve viability and genomic stability. Here, we employed chromosomal reporter constructs to characterize the hierarchy of NHEJ, GC and SSA at a single I-SceI-induced DSB in Chinese hamster ovary cells. We discovered that the use of GC and SSA was increased by 6- to 8-fold upon loss of Ku80 function, suggesting that NHEJ is dominant over the other two pathways. However, NHEJ efficiency was not altered if GC was impaired by Rad51 knockdown. Interestingly, when SSA was made available as an alternative mode for DSB repair, loss of Rad51 function led to an increase in SSA activity at the expense of NHEJ, implying that Rad51 may indirectly promote NHEJ by limiting SSA. We conclude that a repair hierarchy exists to limit the access of the most mutagenic mechanism, SSA, to the break site. Furthermore, the cellular choice of repair pathways is reversible and can be influenced at the level of effector proteins such as Ku80 or Rad51

Saccharomyces cerevisiae-based system for studying clustered DNA damages

DNA-damaging agents can induce clustered lesions or multiply damaged sites (MDSs) on the same or opposing DNA strands. In the latter, attempts to repair MDS can generate closely opposed single-strand break intermediates that may convert non-lethal or mutagenic base damage into double-strand breaks (DSBs). We constructed a diploid S. cerevisiae yeast strain with a chromosomal context targeted by integrative DNA fragments carrying different damages to determine whether closely opposed base damages are converted to DSBs following the outcomes of the homologous recombination repair pathway. As a model of MDS, we studied clustered uracil DNA damages with a known location and a defined distance separating the lesions. The system we describe might well be extended to assessing the repair of MDSs with different compositions, and to most of the complex DNA lesions induced by physical and chemical agents

Cell Cycle-Dependent Induction of Homologous Recombination by a Tightly Regulated I-SceI Fusion Protein

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase

Stimulating fuelwood consumption through public policies: An assessment of economic and resource impacts based on the French Forest Sector Model

International audienceStimulating renewable energy is a crucial objective in view of tackling climate change and coping with future fossil fuel scarcity. In France, fuelwood appears to be an important source for the renewable energy mix. Using the French Forest Sector Model, our paper aims to assess the impacts of three policy options to stimulate fuelwood consumption: a consumer subsidy, a producer subsidy and a fixed-demand contract policy. We explored their impacts in terms of five groups of criteria: (1) forest resource dynamics; (2) variations in wood products prices and quantities consumed and produced; (3) trade balance; (4) budgetary costs; and (5) variations in agent surpluses. We show that no policy option is more desirable than another on the basis of all of these criteria and that trade-offs will determine which is the best policy option to be implemented. © 2013 Elsevier Ltd

Analysis of intrachromosomal homologous recombination in mammalian cell, using tandem repeat sequences

International audienceIn all the organisms, homologous recombination HR is involved in fundamental processes such as genome diversification and DNA repair. Several strategies can be devised to measure homologous recombination in mammalian cells. We present here the interest of using intrachromosomal tandem repeat sequences to measure HR in mammalian cells and we discuss the differences with the ectopic plasmids recombination. The present review focuses on the molecular mechanisms of HR between tandem repeats in mammalian cells. The possibility to use two different orientations of tandem repeats direct or. inverted repeats in parallel constitutes also an advantage. While inverted repeats measure only events arising by strand exchange gene conversion and crossing over , direct repeats monitor strand exchange events and also non-conservative processes such as single strand annealing or replication slippage. In yeast, these processes depend on different pathways, most of them also existing in mammalian cells. These data permit to devise substrates adapted to specific questions about HR in mammalian cells. The effect of substrate structures heterologies, insertionsrdeletions, GT repeats, transcription and consequences of DNA double strand breaks induced by ionizing radiation or endonuclease especially the rare-cutting. endonuclease ISce-I on HR are discussed. Finally, transgenic mouse models using tandem repeats are briefly presented

Visible Light-Driven Electron Transfer from a Dye-Sensitized p-Type NiO Photocathode to a Molecular Catalyst in Solution: Toward NiO-Based Photoelectrochemical Devices for Solar Hydrogen Production

International audienceThe photoelectrochemical activity of a mesoporous NiO electrode sensitized by a ruthenium complex was investigated with several rhodium and cobalt H-2-evolving catalysts. Photocurrent as high as 80 mu A/cm(2) was produced by irradiation of such photocathode in the presence of the Rh(III) polypyridyl complexes, while cobalt complexes gave almost no photocurrent. Photolysis experiments led to the two-electron reduced form of the Rh(III) complexes into Rh(I) complexes and demonstrate the occurrence of an electron transfer chain from NiO to the catalyst. Mott-Schottky experiments evidenced the pH dependence of the NiO flat band potential, explaining the dramatic drop of the photocurrent in acidic conditions (cyanoanilinium). By contrast, in weaker acid conditions (formic acid) the photocurrent increases and the key Rh(III) hydride intermediate was efficiently generated. In acetonitrile solution, Rh(III)-H slowly reacts with HCOOH to generate H-2. However, this process was not catalytic, because the reduction potential of the Ru sensitizer is not sufficiently negative to reduce the Rh(III)-H into Rh(II)-H