Drosophila Xrcc2 regulates DNA double-strand repair in somatic cells.

Genomic integrity is challenged by endo- and exogenous assaults that are combated by highly conserved DNA repair mechanisms. Repair of DNA double-strand breaks (DSBs) is of particular importance, as DSBs inflict chromosome breaks that are the basis of genomic instability. High fidelity recombination repair of DSBs relies on the Rad51 recombinase, aided by several Rad51 paralogs. Despite their significant contribution to DSB repair, the individual roles for Rad51 paralogs are incompletely understood. Drosophila serves as a metazoan model for DNA damage repair at the organismal level. Yet, only two out of four Rad51 paralogs have been studied so far and both are restricted to meiotic recombination repair. Using CRISPR/Cas9 technology, we have generated the first X-ray repair cross complementing 2 (xrcc2) null mutant in Drosophila. Like any other Drosophila Rad51 homologue, loss of xrcc2 does not affect fly development. We found that Drosophila xrcc2 - despite a specific expression in ovaries - is not essential for meiotic DSB repair, but supports the process. In contrast, xrcc2 is required for mitotic DNA damage repair: the mutants are highly sensitive towards various genotoxic stressors, including ionizing radiation, which significantly increase mortality. Moreover, loss of xrcc2 provokes chromosome aberrations in mitotic larval neuroblasts under unstressed conditions and enduring chromosomal breaks as well as persistent repair foci after irradiation exposure. Together these results demonstrate that xrcc2 plays a crucial role in combating genotoxic insult by controlling DSB repair in somatic cells of Drosophila.

Overexpression of the Drosophila ATR homologous checkpoint kinase Mei-41 induces a G2/M checkpoint in Drosophila imaginal tissue.

BACKGROUND: DNA damage generally results in the activation of ATM/ATR kinases and the downstream checkpoint kinases Chk1/Chk2. In Drosophila melanogaster, the ATR homologue meiotic 41 (mei-41) is pivotal to DNA damage repair and cell cycle checkpoint signalling. Although various mei-41 mutant alleles have been analyzed in the past, no gain-of-function allele is yet available. To fill this gap, we have generated transgenic flies allowing temporal and tissue-specific induction of mei-41. RESULTS: Overexpression of mei-41 in wing and eye anlagen affects proliferation and a G2/M checkpoint even in the absence of genomic stress. Similar consequences were observed following the overexpression of the downstream kinase Grapes (Grp) but not of Loki (Lok), encoding the respective Drosophila Chk1 and Chk2 homologues, in agreement with their previously reported activities. Moreover, we show that irradiation induced cell cycle arrest was prolonged in the presence of ectopic mei-41 expression. Similar to irradiation stress, mei-41 triggered the occurrence of a slower migrating form of Grp, implying specific phosphorylation of Grp in response to either signal. Using a p53R-GFP biosensor, we further show that overexpression of mei-41 was sufficient to elicit a robust p53 activation in vivo. CONCLUSION: We conclude that overexpression of the Drosophila ATR homologue mei-41 elicits an effectual DNA damage response irrespective of irradiation.

p53 and cyclin G cooperate in mediating genome stability in somatic cells of Drosophila.

One of the key players in genome surveillance is the tumour suppressor p53 mediating the adaptive response to a multitude of stress signals. Here we identify Cyclin G (CycG) as co-factor of p53-mediated genome stability. CycG has been shown before to be involved in double-strand break repair during meiosis. Moreover, it is also important for mediating DNA damage response in somatic tissue. Here we find it in protein complexes together with p53, and show that the two proteins interact physically in vitro and in vivo in response to ionizing irradiation. In contrast to mammals, Drosophila Cyclin G is no transcriptional target of p53. Genetic interaction data reveal that p53 activity during DNA damage response requires the presence of CycG. Morphological defects caused by overexpression of p53 are ameliorated in cycG null mutants. Moreover, using a p53 biosensor we show that p53 activity is impeded in cycG mutants. As both p53 and CycG are likewise required for DNA damage repair and longevity we propose that CycG plays a positive role in mediating p53 function in genome surveillance of Drosophila.

Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors.

Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. Here we identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. We demonstrate that at low µM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, we show that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations. These findings suggest a new potential role for the PAMAM dendrimers as effective polyvalent channel-blocking inhibitors, which can protect human target cells from intoxication with binary toxins from pathogenic bacteria.