Replication and Recombination Factors Contributing to Recombination-Dependent Bypass of DNA Lesions by Template Switch

Damage tolerance mechanisms mediating damage-bypass and gap-filling are crucial for genome integrity. A major damage tolerance pathway involves recombination and is referred to as template switch. Template switch intermediates were visualized by 2D gel electrophoresis in the proximity of replication forks as X-shaped structures involving sister chromatid junctions. The homologous recombination factor Rad51 is required for the formation/stabilization of these intermediates, but its mode of action remains to be investigated. By using a combination of genetic and physical approaches, we show that the homologous recombination factors Rad55 and Rad57, but not Rad59, are required for the formation of template switch intermediates. The replication-proficient but recombination-defective rfa1-t11 mutant is normal in triggering a checkpoint response following DNA damage but is impaired in X-structure formation. The Exo1 nuclease also has stimulatory roles in this process. The checkpoint kinase, Rad53, is required for X-molecule formation and phosphorylates Rad55 robustly in response to DNA damage. Although Rad55 phosphorylation is thought to activate recombinational repair under conditions of genotoxic stress, we find that Rad55 phosphomutants do not affect the efficiency of X-molecule formation. We also examined the DNA polymerase implicated in the DNA synthesis step of template switch. Deficiencies in translesion synthesis polymerases do not affect X-molecule formation, whereas DNA polymerase δ, required also for bulk DNA synthesis, plays an important role. Our data indicate that a subset of homologous recombination factors, together with DNA polymerase δ, promote the formation of template switch intermediates that are then preferentially dissolved by the action of the Sgs1 helicase in association with the Top3 topoisomerase rather than resolved by Holliday Junction nucleases. Our results allow us to propose the choreography through which different players contribute to template switch in response to DNA damage and to distinguish this process from other recombination-mediated processes promoting DNA repair

Purinergic signalling and immune cells

This review article provides a historical perspective on the role of purinergic signalling in the regulation of various subsets of immune cells from early discoveries to current understanding. It is now recognised that adenosine 5'-triphosphate (ATP) and other nucleotides are released from cells following stress or injury. They can act on virtually all subsets of immune cells through a spectrum of P2X ligand-gated ion channels and G protein-coupled P2Y receptors. Furthermore, ATP is rapidly degraded into adenosine by ectonucleotidases such as CD39 and CD73, and adenosine exerts additional regulatory effects through its own receptors. The resulting effect ranges from stimulation to tolerance depending on the amount and time courses of nucleotides released, and the balance between ATP and adenosine. This review identifies the various receptors involved in the different subsets of immune cells and their effects on the function of these cells

Kernel embedded nonlinear observational mappings in the variational mapping particle filter

Recently, some works have suggested methods to combine variational probabilistic inference withMonte Carlo sampling. One promis- ing approach is via local optimal transport. In this approach, a gradient steepest descent method based on local optimal transport principles is formulated to transform deterministically point samples from an interme- diate density to a posterior density. The local mappings that transform the intermediate densities are embedded in a reproducing kernel Hilbert space (RKHS). This variational mapping method requires the evaluation of the log-posterior density gradient and therefore the adjoint of the ob- servational operator. In this work, we evaluate nonlinear observational mappings in the variational mapping method using two approximations that avoid the adjoint, an ensemble based approximation in which the gradient is approximated by the particle covariances in the state and observational spaces the so-called ensemble space and an RKHS approx- imation in which the observational mapping is embedded in an RKHS and the gradient is derived there. The approximations are evaluated for highly nonlinear observational operators and in a low-dimensional chaotic dynamical system. The RKHS approximation is shown to be highly successful and superior to the ensemble approximation