Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation.

Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair.

Torsional regulation of hRPA-induced unwinding of double-stranded DNA.

All cellular single-stranded (ss) DNA is rapidly bound and stabilized by single stranded DNA-binding proteins (SSBs). Replication protein A, the main eukaryotic SSB, is able to unwind double-stranded (ds) DNA by binding and stabilizing transiently forming bubbles of ssDNA. Here, we study the dynamics of human RPA (hRPA) activity on topologically constrained dsDNA with single-molecule magnetic tweezers. We find that the hRPA unwinding rate is exponentially dependent on torsion present in the DNA. The unwinding reaction is self-limiting, ultimately removing the driving torsional stress. The process can easily be reverted: release of tension or the application of a rewinding torque leads to protein dissociation and helix rewinding. Based on the force and salt dependence of the in vitro kinetics we anticipate that the unwinding reaction occurs frequently in vivo. We propose that the hRPA unwinding reaction serves to protect and stabilize the dsDNA when it is structurally destabilized by mechanical stresses.

Ostreolysin enhances fruiting initiation in the oyster mushroom (Pleurotus ostreatus).

Fruiting initiation in mushrooms can be triggered by a variety of environmental and biochemical stimuli, including substances of natural or synthetic origin. In this work ostreolysin, a cytolytic protein specifically expressed during the formation of primordia and fruit bodies of Pleurotus ostreatus, was applied to nutrient media inoculated with mycelium of P. ostreatus, and its effects on mycelial growth and fructification of the mushroom studied. The addition of ostreolysin slightly inhibited the growth of mycelium, but strongly induced the formation of primordia, which appeared 10 d earlier than in control plates supplemented with bovine serum albumin or with the dissolving buffer alone. Moreover, ostreolysin stimulated the subsequent development of primordia into fruit bodies. However, direct involvement of this protein in the sporulation of the mushroom is unlikely, as it was also detected in large amounts in the non-sporulating strain of P. ostreatus.

Temporal and spatial expression of ostreolysin during development of the oyster mushroom (Pleurotus ostreatus).

In the mushrooms Pleurotus ostreatus and Agrocybe aegerita, expression of the hemolytic proteins ostreolysin and aegerolysin, which belong to the aegerolysin family, has been shown to be initiated specifically during formation of primordia and fruiting bodies. We used rabbit anti-ostreolysin and fluorescent rhodamine-labelled secondary goat antibodies for monitoring ostreolysin and aegerolysin in situ during the mushrooms' development. In parallel, the protein level in developing tissues was monitored with SDS-PAGE and hemolytic assay. Immunolocalization of ostreolysin, visualized by epifluorescence, together with biochemical tests, confirmed specific expression of ostreolysin and aegerolysin in the primordia and fruiting bodies but not in the vegetative mycelia. In the primordia, the proteins were disposed diffusely. In growing and mature fruit bodies they persisted in the lower part of the pileus, in particular in basidia and basidiospores, while in other parts only a few focal remains were observed. Confocal microscopy of immunolabelled sections showed that intracellular ostreolysin was located specifically along the inner edges of hyphae. Since both proteins were found preferentially in the rapidly growing primordia, and in the basidia and basidiospores of maturing fruit bodies, it is suggested that they might play a role in the processes of fructification and/or sporulation.