Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair.

Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3'-protruding ends of a DNA double-strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non-homologous end-joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro-homology (MH) base pair and one nascent base pair. This structure reveals how the N-terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site-directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.

Structures of intermediates along the catalytic cycle of terminal deoxynucleotidyltransferase: dynamical aspects of the two-metal ion mechanism.

Terminal deoxynucleotidyltransferase (Tdt) is a non-templated eukaryotic DNA polymerase of the polX family that is responsible for the random addition of nucleotides at the V(D)J junctions of immunoglobulins and T-cell receptors. Here we describe a series of high-resolution X-ray structures that mimic the pre-catalytic state, the post-catalytic state and a competent state that can be transformed into the two other ones in crystallo via the addition of dAMPcPP and Zn(2+), respectively. We examined the effect of Mn(2+), Co(2+) and Zn(2+) because they all have a marked influence on the kinetics of the reaction. We demonstrate a dynamic role of divalent transition metal ions bound to site A: (i) Zn(2+) (or Co(2+)) in Metal A site changes coordination from octahedral to tetrahedral after the chemical step, which explains the known higher affinity of Tdt for the primer strand when these ions are present, and (ii) metal A has to leave to allow the translocation of the primer strand and to clear the active site, a typical feature for a ratchet-like mechanism. Except for Zn(2+), the sugar puckering of the primer strand 3' terminus changes from C2'-endo to C3'-endo during catalysis. In addition, our data are compatible with a scheme where metal A is the last component that binds to the active site to complete its productive assembly, as already inferred in human pol beta. The new structures have potential implications for modeling pol mu, a closely related polX implicated in the repair of DNA double-strand breaks, in a complex with a DNA synapsis.

Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region.

Terminal deoxynucleotidyltransferase (Tdt) and DNA polymerase mu (pol mu) are two eukaryotic highly similar proteins involved in DNA processing and repair. Despite their high sequence identity, they differ widely in their activity: pol mu has a templated polymerase activity, whereas Tdt has a non-templated one. Loop1, first described when the Tdt structure was solved, has been invoked as the major structural determinant of this difference. Here we describe attempts to transform Tdt into pol mu with the minimal number of mutations in and around Loop1. First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co(++) restores most of this Tdt activity. However, a deletion of the entire Loop1 as in pol lambda does confer a limited template-dependent polymerase behavior to Tdt while a chimera bearing an extended pol mu Loop1 reproduces pol mu behavior. Finally, 16 additional substitutions are reported, targeted at the two so-called 'sequence determinant' regions located just after Loop1 or underneath. Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co(++) addition. These results are discussed in light of the available crystal structures.

ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity.

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using flotation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6.CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.

Similar functional activity of dendritic cells recruited to the mesenteric lymph nodes of newborn and adult mice after the rectal delivery of Mycobacterium bovis BCG.

BCG rectal administration to newborn and adult mice induced protective immune responses against tuberculosis. BCG reaches the sub-epithelial site and the draining mesenteric lymph nodes (MLNs), and dendritic cells (DC) could be recruited to these sites. Using polarized Caco-2 epithelial cells, we showed that BCG translocates epithelial cells to basolateral compartment. Delayed in newborn BALB/c mice, an important recruitment of CD11c+ DCs, was documented in the rectal lamina propria and the MLNs during the first two weeks after rectal BCG delivery. In MLNs, two major DC subtypes were observed: conventional DCs (cDCs) (B220-) and plasmacytoid DCs (pDCs) (B220+). CIRE, mouse DC-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) is predominantly expressed on pDCs and at a higher level on pDCs from the adult compared to newborn MLNs. cDCs with a higher capacity to induce the proliferation of naïve CD4+ T cells than pDCs, triggered CD4+ T cells to produce interferon-gamma whereas pDCs triggered them to release interleukin-10. Both DC subtypes equilibrates T cells as a source of microbicidal/microbiostatic signals and those acting as source of counter-inflammatory signals, preventing tissue damage and/or accelerating tissue repair. Thus, rectal delivery of BCG could be a safe and efficient route of vaccination against tuberculosis.