The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair.

Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.

Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.

MitoStores: chaperone-controlled protein granules store mitochondrial precursors in the cytosol.

Hundreds of nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. However, the early processes associated with mitochondrial protein targeting remain poorly understood. Here, we show that in Saccharomyces cerevisiae, the cytosol has the capacity to transiently store mitochondrial matrix-destined precursors in dedicated deposits that we termed MitoStores. Competitive inhibition of mitochondrial protein import via clogging of import sites greatly enhances the formation of MitoStores, but they also form during physiological cell growth on nonfermentable carbon sources. MitoStores are enriched for a specific subset of nucleus-encoded mitochondrial proteins, in particular those containing N-terminal mitochondrial targeting sequences. Our results suggest that MitoStore formation suppresses the toxic potential of aberrantly accumulating mitochondrial precursor proteins and is controlled by the heat shock proteins Hsp42 and Hsp104. Thus, the cytosolic protein quality control system plays an active role during the early stages of mitochondrial protein targeting through the coordinated and localized sequestration of mitochondrial precursor proteins.

Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts.

DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.

The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria.

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER mitochondria encounter structure (ERMES) and Tom70, together with Djp1 and Lam6, are part of two parallel and partially redundant ER-to-mitochondria delivery routes. When ER-to-mitochondria transfer is prevented by loss of these two contact sites, many precursors of mitochondrial inner membrane proteins are left stranded on the ER membrane, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding.

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

Fast and global reorganization of the chloroplast protein biogenesis network during heat acclimation.

Photosynthesis is a central determinant of plant biomass production, but its homeostasis is increasingly challenged by heat. Little is known about the sensitive regulatory principles involved in heat acclimation that underly the biogenesis and repair of chloroplast-encoded core subunits of photosynthetic complexes. Employing time-resolved ribosome and transcript profiling together with selective ribosome proteomics, we systematically deciphered these processes in chloroplasts of Chlamydomonas reinhardtii. We revealed protein biosynthesis and altered translation elongation as central processes for heat acclimation and showed that these principles are conserved between the alga and the flowering plant Nicotiana tabacum. Short-term heat exposure resulted in specific translational repression of chlorophyll a-containing core antenna proteins of photosystems I and II. Furthermore, translocation of ribosome nascent chain complexes to thylakoid membranes was affected, as reflected by the increased accumulation of stromal cpSRP54-bound ribosomes. The successful recovery of synthesizing these proteins under prolonged acclimation of nonlethal heat conditions was associated with specific changes of the co-translational protein interaction network, including increased ribosome association of chlorophyll biogenesis enzymes and acclimation factors responsible for complex assembly. We hypothesize that co-translational cofactor binding and targeting might be bottlenecks under heat but become optimized upon heat acclimation to sustain correct co-translational protein complex assembly.

CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination.

A key event during eukaryotic replication termination is the removal of the CMG helicase from chromatin. CMG unloading involves ubiquitylation of its Mcm7 subunit and the action of the p97 ATPase. Using a proteomic screen in Xenopus egg extracts, we identified factors that are enriched on chromatin when CMG unloading is blocked. This approach identified the E3 ubiquitin ligase CRL2Lrr1, a specific p97 complex, other potential regulators of termination, and many replisome components. We show that Mcm7 ubiquitylation and CRL2Lrr1 binding to chromatin are temporally linked and occur only during replication termination. In the absence of CRL2Lrr1, Mcm7 is not ubiquitylated, CMG unloading is inhibited, and a large subcomplex of the vertebrate replisome that includes DNA Pol ε is retained on DNA. Our data identify CRL2Lrr1 as a master regulator of replisome disassembly during vertebrate DNA replication termination.

Single-cell transcription profiles in Bloom syndrome patients link BLM deficiency with altered condensin complex expression signatures.

Bloom syndrome (BS) is an autosomal recessive disease clinically characterized by primary microcephaly, growth deficiency, immunodeficiency and predisposition to cancer. It is mainly caused by biallelic loss-of-function mutations in the BLM gene, which encodes the BLM helicase, acting in DNA replication and repair processes. Here, we describe the gene expression profiles of three BS fibroblast cell lines harboring causative, biallelic truncating mutations obtained by single-cell (sc) transcriptome analysis. We compared the scRNA transcription profiles from three BS patient cell lines to two age-matched wild-type controls and observed specific deregulation of gene sets related to the molecular processes characteristically affected in BS, such as mitosis, chromosome segregation, cell cycle regulation and genomic instability. We also found specific upregulation of genes of the Fanconi anemia pathway, in particular FANCM, FANCD2 and FANCI, which encode known interaction partners of BLM. The significant deregulation of genes associated with inherited forms of primary microcephaly observed in our study might explain in part the molecular pathogenesis of microcephaly in BS, one of the main clinical characteristics in patients. Finally, our data provide first evidence of a novel link between BLM dysfunction and transcriptional changes in condensin complex I and II genes. Overall, our study provides novel insights into gene expression profiles in BS on an sc level, linking specific genes and pathways to BLM dysfunction.

Mechanism of replication-coupled DNA interstrand crosslink repair.

DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on Xenopus egg extracts that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase zeta. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.

The versatile interactome of chloroplast ribosomes revealed by affinity purification mass spectrometry.

In plant cells, chloroplast gene expression is predominantly controlled through post-transcriptional regulation. Such fine-tuning is vital for precisely orchestrating protein complex assembly as for the photosynthesis machinery and for quickly responding to environmental changes. While regulation of chloroplast protein synthesis is of central importance, little is known about the degree and nature of the regulatory network, mainly due to challenges associated with the specific isolation of transient ribosome interactors. Here, we established a ribosome affinity purification method, which enabled us to broadly uncover putative ribosome-associated proteins in chloroplasts. Endogenously tagging of a protein of the large or small subunit revealed not only interactors of the holo complex, but also preferential interactors of the two subunits. This includes known canonical regulatory proteins as well as several new proteins belonging to the categories of protein and RNA regulation, photosystem biogenesis, redox control and metabolism. The sensitivity of the here applied screen was validated for various transiently interacting proteins. We further provided evidence for the existence of a ribosome-associated Nα-acetyltransferase in chloroplasts and its ability to acetylate substrate proteins at their N-terminus. The broad set of ribosome interactors underscores the potential to regulate chloroplast gene expression on the level of protein synthesis.

Electroneutral Polymer Nanodiscs Enable Interference-Free Probing of Membrane Proteins in a Lipid-Bilayer Environment.

Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent-solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral-yet water-soluble-polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo-DIBMA and Sulfo-SMA(2:1). Sulfo-DIBMA and Sulfo-SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein-lipid interactions through lab-on-a-chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs.

XPF-ERCC1 acts in Unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4.

DNA interstrand crosslinks (ICLs), highly toxic lesions that covalently link the Watson and Crick strands of the double helix, are repaired by a complex, replication-coupled pathway in higher eukaryotes. The earliest DNA processing event in ICL repair is the incision of parental DNA on either side of the ICL ("unhooking"), which allows lesion bypass. Incisions depend critically on the Fanconi anemia pathway, whose activation involves ubiquitylation of the FANCD2 protein. Using Xenopus egg extracts, which support replication-coupled ICL repair, we show that the 3' flap endonuclease XPF-ERCC1 cooperates with SLX4/FANCP to carry out the unhooking incisions. Efficient recruitment of XPF-ERCC1 and SLX4 to the ICL depends on FANCD2 and its ubiquitylation. These data help define the molecular mechanism by which the Fanconi anemia pathway promotes a key event in replication-coupled ICL repair.

The intermembrane space protein Mix23 is a novel stress-induced mitochondrial import factor.

The biogenesis of mitochondria requires the import of hundreds of precursor proteins. These proteins are transported post-translationally with the help of chaperones, meaning that the overproduction of mitochondrial proteins or the limited availability of chaperones can lead to the accumulation of cytosolic precursor proteins. This imposes a severe challenge to cytosolic proteostasis and triggers a specific transcription program called the mitoprotein-induced stress response, which activates the proteasome system. This coincides with the repression of mitochondrial proteins, including many proteins of the intermembrane space. In contrast, herein we report that the so-far-uncharacterized intermembrane space protein Mix23 is considerably up-regulated when mitochondrial import is perturbed. Mix23 is evolutionarily conserved and a homolog of the human protein CCDC58. We found that, like the subunits of the proteasome, Mix23 is under control of the transcription factor Rpn4. It is imported into mitochondria by the mitochondrial disulfide relay. Mix23 is critical for the efficient import of proteins into the mitochondrial matrix, particularly if the function of the translocase of the inner membrane 23 is compromised such as in temperature-sensitive mutants of Tim17. Our observations identify Mix23 as a novel regulator or stabilizer of the mitochondrial protein import machinery that is specifically up-regulated upon mitoprotein-induced stress conditions.

Homology-driven assembly of NOn-redundant protEin sequence sets (NOmESS) for mass spectrometry.

UNLABELLED: To enable mass spectrometry (MS)-based proteomic studies with poorly characterized organisms, we developed a computational workflow for the homology-driven assembly of a non-redundant reference sequence dataset. In the automated pipeline, translated DNA sequences (e.g. ESTs, RNA deep-sequencing data) are aligned to those of a closely related and fully sequenced organism. Representative sequences are derived from each cluster and joined, resulting in a non-redundant reference set representing the maximal available amino acid sequence information for each protein. We here applied NOmESS to assemble a reference database for the widely used model organism Xenopus laevis and demonstrate its use in proteomic applications. AVAILABILITY AND IMPLEMENTATION: NOmESS is written in C#. The source code as well as the executables can be downloaded from http://www.biochem.mpg.de/cox Execution of NOmESS requires BLASTp and cd-hit in addition. CONTACT: cox@biochem.mpg.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Distinct types of intramitochondrial protein aggregates protect mitochondria against proteotoxic stress.

Mitochondria consist of hundreds of proteins, most of which are inaccessible to the proteasomal quality control system of the cytosol. How cells stabilize the mitochondrial proteome during challenging conditions remains poorly understood. Here, we show that mitochondria form spatially defined protein aggregates as a stress-protecting mechanism. Two different types of intramitochondrial protein aggregates can be distinguished. The mitoribosomal protein Var1 (uS3m) undergoes a stress-induced transition from a soluble, chaperone-stabilized protein that is prevalent under benign conditions to an insoluble, aggregated form upon acute stress. The formation of Var1 bodies stabilizes mitochondrial proteostasis, presumably by sequestration of aggregation-prone proteins. The AAA chaperone Hsp78 is part of a second type of intramitochondrial aggregate that transiently sequesters proteins and promotes their folding or Pim1-mediated degradation. Thus, mitochondrial proteins actively control the formation of distinct types of intramitochondrial protein aggregates, which cooperate to stabilize the mitochondrial proteome during proteotoxic stress conditions.

TopBP1 utilises a bipartite GINS binding mode to support genome replication.

Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Polε subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.

Characterization of a soluble library of the Pseudomonas aeruginosa PAO1 membrane proteome with emphasis on c-di-GMP turnover enzymes.

Studies of protein-protein interactions in membranes are very important to fully understand the biological function of a cell. The extraction of proteins from the native membrane environment is a critical step in the preparation of membrane proteins that might affect the stability of protein complexes. In this work, we used the amphiphilic diisobutylene/maleic acid copolymer to extract the membrane proteome of the opportunistic pathogen Pseudomonas aeruginosa, thereby creating a soluble membrane-protein library within a native-like lipid-bilayer environment. Size fractionation of nanodisc-embedded proteins and subsequent mass spectrometry enabled the identification of 3358 proteins. The native membrane-protein library showed a very good overall coverage compared to previous proteome data. The pattern of size fractionation indicated that protein complexes were preserved in the library. More than 20 previously described complexes, e.g. the SecYEG and Pili complexes, were identified and analyzed for coelution. Although the mass-spectrometric dataset alone did not reveal new protein complexes, combining pulldown assays with mass spectrometry was successful in identifying new protein interactions in the native membrane-protein library. Thus, we identified several candidate proteins for interactions with the membrane phosphodiesterase NbdA, a member of the c-di-GMP network. We confirmed the candidate proteins CzcR, PA4200, SadC, and PilB as novel interaction partners of NbdA using the bacterial adenylate cyclase two-hybrid assay. Taken together, this work demonstrates the usefulness of the native membrane-protein library of P. aeruginosa for the investigation of protein interactions and membrane-protein complexes. Data are available via ProteomeXchange with identifiers PXD039702 and PXD039700.

The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import.

Mitochondria play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here, we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenic enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and increases the levels of its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in compromised protein import into mitochondria and reduced steady-state levels of mitochondrial proteins. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery to improve mitochondrial protein import during phases of metabolic transition.

The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.