CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication

Structural insights into Omp85-mediated protein translocation and insertion in the bacterial outer membrane

Proteins of the Omp85 superfamily reside in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts and are responsible for the insertion of outer membrane β-barrel proteins into or the translocation of soluble proteins across the membrane. They contain a C-terminal membrane-embedded 16-stranded β-barrel and soluble substrate-interacting POTRA domains, which in Gram-negative bacteria locate to the periplasm. The underlying translocation and insertion mechanisms are poorly understood and atomic structures of Omp85 insertases have been missing. This thesis provides the structural basis for the insertion mechanism of substrates by the Escherichia coli Omp85 insertase TamA. The crystal structure of TamA reveals minimal interactions between the first and the last β-strand of the barrel with a lipid-occupied lateral gate, suggesting substrate β-barrel assembly via hybrid barrel formation and lateral release. Exemplified by the crystallization of TamA, a general crystal seeding protocol for optimization of membrane protein crystals grown from bicelle solution is described. Furthermore, interactions of TamA with its associated periplasmic complex partner TamB are investigated by experimental approaches and bioinformatics, revealing potential interaction sites between these two proteins. The organization of Omp85 insertases is then compared to Omp85 translocases, represented by FhaC from Bordetella pertussis, and a mechanism for substrate selection by FhaC is deduced from a newly determined crystal structure of an FhaC double mutant defective in substrate recognition. Whereas protein import into chloroplasts is mediated by a member of the Omp85 superfamily, in mitochondria this task is fulfilled by Tom40, a 19-stranded β-barrel outer membrane protein that lacks POTRA domains. As a basis for experimental in vitro approaches to gaining insights into the Tom40 translocation mechanism, a protocol for recombinant Tom40 over-expression, refolding and sample preparation is provided. NMR spectroscopy of isotope-labeled protein evidences the presence of folded Tom40 in our samples

Conserved Omp85 lid-lock structure and substrate recognition in FhaC

Omp85 proteins mediate translocation of polypeptide substrates across and into cellular membranes. They share a common architecture comprising substrate-interacting POTRA domains, a C-terminal 16-stranded β-barrel pore and two signature motifs located on the inner barrel wall and at the tip of the extended L6 loop. The observation of two distinct conformations of the L6 loop in the available Omp85 structures previously suggested a functional role of conformational changes in L6 in the Omp85 mechanism. Here we present a 2.5 Å resolution structure of a variant of the Omp85 secretion protein FhaC, in which the two signature motifs interact tightly and form the conserved 'lid lock'. Reanalysis of previous structural data shows that L6 adopts the same, conserved resting state position in all available Omp85 structures. The FhaC variant structure further reveals a competitive mechanism for the regulation of substrate binding mediated by the linker to the N-terminal plug helix H1

Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA

TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family

Mutations in the voltage-sensing domain affect the alternative ion permeation pathway in the TRPM3 channel

KEY POINTS: Mutagenesis at positively charged amino acids (arginines and lysines) (R1-R4) in the voltage-sensor domain (transmembrane segment (S) 4) of voltage-gated Na+ , K+ and Ca2+ channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non-canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non-canonical pore exists in the native TRPM3 channel and can be activated by co-stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K+ channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage-sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non-canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage-sensing domain of TRPM3 in the formation of an alternative ion permeation pathway. ABSTRACT: Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1-S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K+ channels, in which mutations in S4 can create an analogous 'omega' pore, we performed site-directed mutagenesis studies and patch clamp experiments to identify amino acid residues involved in the formation of the non-canonical pore in TRPM3. Based on our results, we pinpoint four residues in S4 (W982, R985, D988 and G991) as crucial determinants of the properties of the alternative ion permeation pathway.status: publishe

Generic and Automated Drive-by GPU Cache Attacks from the Browser

International audienceIn recent years, the use of GPUs for general-purpose computationshas steadily increased. As security-critical computations like AESare becoming more common on GPUs, the scrutiny must also in-crease. At the same time, new technologies like WebGPU put easyaccess to compute shaders in every web browser. Prior work hasshown that GPU caches are vulnerable to the same eviction-basedattacks as CPUs, e.g., Prime+Probe, from native code.In this paper, we present the first GPU cache side-channel attackfrom within the browser, more specifically from the restricted We-bGPU environment. The foundation for our generic and automatedattacks are self-configuring primitives applicable to a wide varietyof devices, which we demonstrate on a set of 11 desktop GPUsfrom 5 different generations and 2 vendors. We leverage featuresof the new WebGPU standard to create shaders that implement allbuilding blocks needed for cache side-channel attacks, such as tech-niques to distinguish L2 cache hits from misses. Beyond the stateof the art, we leverage the massive parallelism of modern GPUsto design the first parallelized eviction set construction algorithm.Based on our attack primitives, we present three case studies: First,we present an inter-keystroke timing attack with high F1-scores,i.e., 82 % to 98 % on NVIDIA. Second, we demonstrate a generic,set-agnostic, end-to-end attack on a GPU-based AES encryptionservice, leaking a full AES key in 6 minutes. Third, we evaluate anative-to-browser data-exfiltration scenario with a Prime+Probecovert channel that achieves transmission rates of up to 10.9 kB/s.Our attacks require no user interaction and work in a time framethat easily enables drive-by attacks while browsing the Internet.Our work emphasizes that browser vendors need to treat access tothe GPU similar to other security- and privacy-related resources

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.</p