Aerobic anoxygenic phototrophs (AAPs) are prokaryotic microorganisms capable of harvesting light using bacteriochlorophyll-based reaction centres. Marine AAP communities are generally dominated by species belonging to the Roseobacter clade. For this reason, we used marine Roseobacter-related strain COL2P as a model organism to characterize its photosynthetic apparatus, level of pigmentation and expression of photosynthetic complexes. This strain contained functional photosynthetic reaction centres with bacteriochlorophyll a and spheroidenone as the main light-harvesting pigments, but the expression of the photosynthetic apparatus was significantly reduced when compared to truly photoautotrophic species. Moreover, the absence of peripheral light-harvesting complexes largely reduced its light-harvesting capacity. The size of the photosynthetic unit was limited to 35.4 +/- 1.0 BChl a molecules supplemented by the same number of spheroidenone molecules. The contribution of oxidative phosphorylation and photophosphorylation was analysed by respiration and fluorometric measurements. Our results indicate that even with a such reduced photosynthetic apparatus, photophosphorylation provides up to three times higher electron fluxes than aerobic respiration. These results suggest that light-derived energy can provide a substantial fraction of COL2P metabolic needs.
Fanconi anemia (FA) is a genetic disorder characterized by a defect in DNA interstrand crosslink (ICL) repair, chromosomal instability, and a predisposition to cancer. Recently, two RAD51 mutations were reported to cause an FA-like phenotype. Despite the tight association of FA/HR proteins with replication fork (RF) stabilization during normal replication, it remains unknown how FA-associated RAD51 mutations affect replication beyond ICL lesions. Here, we report that these mutations fail to protect nascent DNA from MRE11-mediated degradation during RF stalling in Xenopus laevis egg extracts. Reconstitution of DNA protection in vitro revealed that the defect arises directly due to altered RAD51 properties. Both mutations induce pronounced structural changes and RAD51 filament destabilization that is not rescued by prevention of ATP hydrolysis due to aberrant ATP binding. Our results further interconnect the FA pathway with DNA replication and provide mechanistic insight into the role of RAD51 in recombination-independent mechanisms of genome maintenance.
Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an ‘open’ conformation when compared to a ‘closed’ structure formed by mutants, reflecting alterations in helical pitch. The kinetics of formation/disassembly of RAD51 filaments show rapid and high ssDNA coverage via low cooperativity binding of RAD51 units along the DNA. Subsequently, a series of isomerization or dissociation events mediated by nucleotide binding state creates intrinsically dynamic RAD51 NFs. Our findings highlight important a mechanistic divergence among recombinases from different organisms, in line with the diversity of biological mechanisms of HR initiation and quality control. These data reveal unexpected intrinsic dynamic properties of the RAD51 filament during assembly/disassembly, which may be important for the proper control of homologous recombination.
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