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International audience; HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity.

International audience; LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar Kd determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from 13 additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.

International audience; Retroelements represent a considerable fraction of many eukaryotic genomes and are considered major drives for adaptive genetic innovations. Recent discoveries showed that despite not normally using DNA intermediates like retroviruses do, Mononegaviruses (i.e., viruses with nonsegmented, negative-sense RNA genomes) can integrate gene fragments into the genomes of their hosts. This was shown for Bornaviridae and Filoviridae, the sequences of which have been found integrated into the germ line cells of many vertebrate hosts. Here, we show that Rhabdoviridae sequences, the major Mononegavirales family, have integrated only into the genomes of arthropod species. We identified 185 integrated rhabdoviral elements (IREs) coding for nucleoproteins, glycoproteins, or RNA-dependent RNA polymerases; they were mostly found in the genomes of the mosquito Aedes aegypti and the blacklegged tick Ixodes scapularis. Phylogenetic analyses showed that most IREs in A. aegypti derived from multiple independent integration events. Since RNA viruses are submitted to much higher substitution rates as compared with their hosts, IREs thus represent fossil traces of the diversity of extinct Rhabdoviruses. Furthermore, analyses of orthologous IREs in A. aegypti field mosquitoes sampled worldwide identified an integrated polymerase IRE fragment that appeared under purifying selection within several million years, which supports a functional role in the host's biology. These results show that A. aegypti was subjected to repeated Rhabdovirus infectious episodes during its evolution history, which led to the accumulation of many integrated sequences. They also suggest that like retroviruses, integrated rhabdoviral sequences may participate actively in the evolution of their hosts.

The deposited article is a post-print version and has peer review. The deposited article version contains attached the supplementary materials within the pdf. This publication hasn't any creative commons license associated. Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity. Ligue Nationale contre le Cancer (Equipe labellisée Ligue 2010); PIC Programs; the European Commission Network of Excellence EpiGeneSys (HEALTH-F4-2010-257082), the European Commission ITN FP7-PEOPLE-2007-215148 “Image DDR” and FP7-PEOPLE-2008-238176 “Nucleosome 4D,” ERC Advanced Grant 2009-AdG_20090506 “Eccentric,” the European Commission large-scale integrating project FP7_HEALTH-2010-259743 “MODHEP,” ANR “ECenS” ANR-09-BLAN-0257-01, ANR “ChromaTin” ANR-10-BLAN-1326-03, and INCa “GepiG.” The lab of P.D.A. is funded by NIA program project P01 AG031862. L.E.T.J. was supported by the Fundação Calouste Gulbenkian, Fundação para a Ciência e a Tecnologia (FCT) grant BIA-PRO/100537/2008, the European Commission FP7 programme, and an EMBO installation grant. info:eu-repo/semantics/publishedVersion

International audience; Deinococcus radiodurans contains a highly condensed nucleoid that remains to be unaltered following the exposure to high doses of gamma-irradiation. Proteins belonging to the structural maintenance of chromosome protein (SMC) family are present in all organisms and were shown to be involved in chromosome condensation, pairing, and/or segregation. Here, we have inactivated the smc gene in the radioresistant bacterium D. radiodurans, and, unexpectedly, found that smc null mutants showed no discernible phenotype except an increased sensitivity to gyrase inhibitors suggesting a role of SMC in DNA folding. A defect in the SMC-like SbcC protein exacerbated the sensitivity to gyrase inhibitors of cells devoid of SMC. We also showed that the D. radiodurans SMC protein forms discrete foci at the periphery of the nucleoid suggesting that SMC could locally condense DNA. The phenotype of smc null mutant leads us to speculate that other, not yet identified, proteins drive the compact organization of the D. radiodurans nucleoid.

Orthologs of proteins SbcD (Mre11) and SbcC (Rad50) exist in all kingdoms of life and are involved in a wide variety of DNA repair and maintenance functions, including homologous recombination and nonhomologous end joining. Here, we have inactivated the sbcC and/or sbcD genes of Deinococcus radiodurans , a highly radioresistant bacterium able to mend hundreds of radiation-induced DNA double-strand breaks (DSB). Mutants devoid of the SbcC and/or SbcD proteins displayed reduced survival and presented a delay in kinetics of DSB repair and cell division following γ-irradiation. It has been recently reported that D. radiodurans DNA polymerase X (PolX) possesses a structure-modulated 3′-to-5′ exonuclease activity reminiscent of specific nuclease activities displayed by the SbcCD complex from Escherichia coli . We constructed a double mutant devoid of SbcCD and PolX proteins. The double-mutant Δ sbcCD Δ polX Dr (where Dr indicates D. radiodurans ) bacteria are much more sensitive to γ-irradiation than the single mutants, suggesting that the deinococcal SbcCD and PolX proteins may play important complementary roles in processing damaged DNA ends. We propose that they are part of a backup repair system acting to rescue cells containing DNA lesions that are excessively numerous or difficult to repair.

We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.

International audience; In Bacillus subtilis, expression of the rocG gene, encoding glutamate dehydrogenase, and the rocABC operon, involved in arginine catabolism, requires SigL (sigma(54))-containing RNA polymerase as well as RocR, a positive regulator of the NtrC/NifA family. The RocR protein was purified and shown to bind specifically to the intergenic region located between rocG and the rocABC operon. DNaseI footprinting experiments were used to define the RocR-binding site as an 8 bp inverted repeat, separated by one base pair, forming an imperfect palindrome which is present twice within the rocG-rocABC intergenic region, acting as both a downstream activating sequence (DAS) and an upstream activating sequence (UAS). Point mutations in either of these two sequences significantly lowered expression of both rocG and rocABC. This bidirectional enhancer element retained partial activity even when moved 9 kb downstream of the rocA promoter. Electron microscopy experiments indicated that an intrinsically curved region is located between the UAS/DAS region and the promoter of the rocABC operon. This curvature could facilitate interaction of RocR with sigma(54)-RNA polymerase at the rocABC promoter.

International audience; Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.

International audience