Mechanistic Issues of the Interaction of the Hairpin-Forming Domain of tBid with Mitochondrial Cardiolipin

BACKGROUND: The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated. PRINCIPAL FINDINGS: The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix alphaH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, alphaH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix alphaH6 to efficiently induce cytochrome c release and apoptosis. CONCLUSIONS: Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix alphaH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix alphaH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae

Abstract RecA-mediated Homologous Recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae ( Sp RecA), in comparison to the Escherichia coli RecA ( Ec RecA) paradigm. Ec RecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block Ec RecA nucleofilament growth. We report that neither of the two paralogous pneumococcal SSBs could assist Sp RecA polymerization on ssDNA. Instead, we found that the conserved RadA helicase promotes this Sp RecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native Sp RecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the Ec RecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that Sp RecA and Ec RecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes

Rad51 Polymerization Reveals a New Chromatin Remodeling Mechanism

Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics

Etude par microscopies moléculaires des mécanismes impliqués dans la régulation des étapes précoces et tardives de la recombinaison homologue

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Recombinases and Related Proteins in the Context of Homologous Recombination Analyzed by Molecular Microscopy

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In vitro role of Rad54 in Rad51-ssDNA filament-dependent homology search and synaptic complexes formation.

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54-K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation

Rad54 is critical for homology search and formation of synaptic complexes by the Rad51-ssDNA filament.

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Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy

International audienceDNA intermediate structures are formed in all major pathways of DNA metabolism. Transmission electron microscopy(TEM) is a tool of choice to study their choreography and has led to major advances in the understanding of these mechanisms,particularly those of homologous recombination (HR) and replication. In this article, we describe specific TEM proceduresdedicated to the structural characterization of DNA intermediates formed during these processes. These particularDNA species contain single-stranded DNA regions and/or branched structures, which require controlling both the DNA moleculesspreading and their staining for subsequent visualization using dark-field imaging mode. Combining BAC (benzyl dimethylalkyl ammoniumchloride) film hyperphase with positive staining and dark-field TEM allows characterizing syntheticDNA substrates, joint molecules formed during not only in vitro assays mimicking HR, but also in vivo DNAintermediates

Replication intermediate architecture reveals the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells

Abstract DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remain elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η , able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of pol η in shaping this response and ensuring the continuity of DNA synthesis

Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

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