Functional and structural basis for a bacteriophage homolog of human RAD52.

In eukaryotes, homologous recombination proteins such as RAD51 and RAD52 play crucial roles in DNA repair and genome stability. Human RAD52 is a member of a large single-strand annealing protein (SSAP) family [1] and stimulates Rad51-dependent recombination [2, 3]. In prokaryotes and phages, it has been difficult to establish the presence of RAD52 homologs with conserved sequences. Putative SSAPs were recently found in several phages that infect strains of Lactococcus lactis[4]. One of these SSAPs was identified as Sak and was found in the virulent L. lactis phage ul36, which belongs to the Siphoviridae family [4, 5]. In this study, we show that Sak is homologous to the N terminus of human RAD52. Purified Sak binds single-stranded DNA (ssDNA) preferentially over double-stranded DNA (dsDNA) and promotes the renaturation of long complementary ssDNAs. Sak also binds RecA and stimulates homologous recombination reactions. Mutations shown to modulate RAD52 DNA binding [6] affect Sak similarly. Remarkably, electron-microscopic reconstruction of Sak reveals an undecameric (11) subunit ring, similar to the crystal structure of the N-terminal fragment of human RAD52 [7, 8]. For the first time, we propose a viral homolog of RAD52 at the amino acid, phylogenic, functional, and structural levels.

Effects of (p)ppGpp on the progression of the cell cycle of Caulobacter crescentus.

Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra- or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p)ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p)ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p)ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p)ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p)ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p)ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p)ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.

Remaining rod activity mediates visual behavior in adult Rpe65-/- mice.

Purpose: C57/Bl6, Cpfl1-/- (Cone photoreceptors function loss 1; pure rod function), Gnat1alpha-/- (rod alpha-transducin; pure cone function) and Rpe65-/-;Rho-/- double knock-out mice were studied in order to distinguish the respective contributions of the different photoreceptor (PR) systems that enable light perception and mediate a visual reflex in adult Rpe65-/- mice using a simple behavioural procedure. Methods: Visual function was estimated using a rotating automatized optomotor drum covered with vertical black and white stripes at spatial frequencies of 0.025 to 0.5 cycles per degree (cpd) in both photopic and scotopic conditions. To evaluate the contribution as well as the light intensity threshold of each PR system, we tested the mouse strains with different luminances. Results: Stripe rotation elicits head movements in wild-type (WT) animals in photopic and scotopic conditions depending on the spatial frequency, whereas Cpfl1-/- mice show a reduced activity in the photopic condition and Gnat1alpha-/- mice an almost absent response in the scotopic condition. Interestingly, a robust visual response is obtained with Rpe65-/- knockout mice at 0.075 cpd and 0.1 cpd in the photopic condition. The residual rod function in the Rpe65-/- animals was demonstrated by testing Rpe65-/-;Rho-/- mice that present no response in photopic conditions. Conclusions: The optomotor test is a simple method to estimate the visual function, and to evaluate the respective contributions of rod and cone systems. Using this test, we demonstrate that in Rpe65-/- mice, devoid of functional cones and of detectable 11-cis-retinal protein, rods mimic in part the cone function by mediating vision in photopic conditions.

Interactions between Siglec-7/9 receptors and ligands influence NK cell-dependent tumor immunosurveillance.

Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid-binding Ig-like lectins Siglec-7 and -9 are MHC class I-independent inhibitory receptors on human NK cells that recognize sialic acid-containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9+ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell-sensitive tumor cells and, unexpectedly, of presumably NK cell-resistant tumor cells to NK cell-mediated cytotoxicity. Together, these observations have direct implications for NK cell-based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.