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Replisome collision with a nicked parental DNA template can lead to the formation of a replication-associated double strand break (DSB). How this break is repaired has implications for cancer initiation, cancer therapy and therapeutic gene editing. Recent work shows that collision of a replisome with a nicked DNA template can give rise to either a single-ended (se) or a double-ended (de)DSB, with potentially divergent effects on repair pathway choice and genomic instability. Emerging evidence suggests that the biochemical environment of the broken mammalian replication fork may be specialized in such a way as to skew repair in favor of homologous recombination at the expense of non-homologous end joining., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The E3 ubiquitin ligase TRAIP associates with the replisome and helps this molecular machine deal with replication stress. Thus, TRAIP promotes DNA inter-strand crosslink repair by triggering the disassembly of CDC45-MCM2-7-GINS (CMG) helicases that have converged on these lesions. However, disassembly of single CMGs that have stalled temporarily would be deleterious, suggesting that TRAIP must be carefully regulated. Here, we demonstrate that human cells lacking the de-ubiquitylating enzyme USP37 are hypersensitive to topoisomerase poisons and other replication stress-inducing agents. We further show that TRAIP loss rescues the hypersensitivity of USP37 knockout cells to topoisomerase inhibitors. In Xenopus egg extracts depleted of USP37, TRAIP promotes premature CMG ubiquitylation and disassembly when converging replisomes stall. Finally, guided by AlphaFold-Multimer, we discovered that binding to CDC45 mediates USP37's response to topological stress. In conclusion, we propose that USP37 protects genome stability by preventing TRAIP-dependent CMG unloading when replication stress impedes timely termination., Competing Interests: Competing interests J.C.W. is a co-founder of MOMA Therapeutics, in which he has a financial interest. S.P.J. is Chief Research Officer (part time) at Insmed Innovation UK. Ltd. and founding partner of Ahren Innovation Capital LLP. He is a board member and chair of Scientific Advisory Board of Mission Therapeutics Ltd. and is a consultant and shareholder of Inflex Ltd. The remaining authors declare no competing interests.

Using CRISPR-Cas9 nicking enzymes, we examined the interaction between the replication machinery and single-strand breaks, one of the most common forms of endogenous DNA damage. We show that replication fork collapse at leading-strand nicks generates resected single-ended double-strand breaks (seDSBs) that are repaired by homologous recombination (HR). If these seDSBs are not promptly repaired, arrival of adjacent forks creates double-ended DSBs (deDSBs), which could drive genomic scarring in HR-deficient cancers. deDSBs can also be generated directly when the replication fork bypasses lagging-strand nicks. Unlike deDSBs produced independently of replication, end resection at nick-induced seDSBs and deDSBs is BRCA1-independent. Nevertheless, BRCA1 antagonizes 53BP1 suppression of RAD51 filament formation. These results highlight distinctive mechanisms that maintain replication fork stability.

In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles in vivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2 Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Finally, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates., (© 2024 John Wiley & Sons Ltd.)

In transcription-coupled repair, stalled RNA polymerase II (Pol II) is recognized by CSB and CRL4 CSA , which co-operate with UVSSSA and ELOF1 to recruit TFIIH for nucleotide excision repair (TC-NER). To explore the mechanism of TC-NER, we recapitulated this reaction in vitro . When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4 CSA , UVSSA, and ELOF1. Repair also depends on STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9 Å cryo-electron microscopy structure shows that STK19 joins the TC-NER complex by binding CSA and the RPB1 subunit of Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of Pol II for lesion verification. In summary, our analysis of cell-free TC-NER suggests that STK19 couples RNA polymerase II stalling to downstream repair events., Competing Interests: COMPETING INTEREST STATEMENT J.C.W. is a co-founder of MoMa therapeutics, in which he has a financial interest.

Nucleosomes are the fundamental unit of eukaryotic chromatin. Diverse factors interact with nucleosomes to modulate chromatin architecture and facilitate DNA repair, replication, transcription, and other cellular processes. An important platform for chromatin binding is the H2A-H2B acidic patch. Here, we used AlphaFold-Multimer to screen over 7000 human proteins for nucleosomal acidic patch binding and identify 41 potential acidic patch binders. We determined the cryo-EM structure of one hit, SHPRH, with the nucleosome at 2.8 Å. The structure confirms the predicted acidic patch interaction, reveals that the SHPRH ATPase engages a different nucleosomal DNA location than other SF2-type ATPases, and clarifies the roles of SHPRH's domains in nucleosome recognition. Our results illustrate the use of in silico screening as a high throughput method to identify specific interaction types and expands the set of potential acidic patch binding factors., All the Screening Data Is Freely Available at: https://predictomes.org/view/acidicpatch.

Polycomb (Pc) group proteins are transcriptional regulators with key roles in development, cell identity, and differentiation. Pc-bound chromatin regions form repressive domains that interact in 3D to assemble repressive nuclear compartments. Here, we use multiplexed chromatin imaging to investigate whether Pc compartments involve the clustering of multiple Pc domains during Drosophila development. Notably, 3D proximity between Pc targets is rare and involves predominantly pairwise interactions. These 3D proximities are particularly enhanced in segments where Pc genes are co-repressed. In addition, segment-specific expression of Hox Pc targets leads to their spatial segregation from Pc-repressed genes. Finally, non-Hox Pc targets are more proximal in regions where they are co-expressed. These results indicate that long-range Pc interactions are temporally and spatially regulated during differentiation and development but do not induce frequent clustering of multiple distant Pc genes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ., (© 2024. The Author(s).)

Recent advances in experimental fluorescence microscopy allow high accuracy determination (resolution of 50 nm) of the three-dimensional physical location of multiple (up to ∼10^{2}) tagged regions of the chromosome. We investigate publicly available microscopy data for two loci of the human Chr21 obtained from multiplexed fluorescence in situ hybridization (FISH) methods for different cell lines and treatments. Inspired by polymer physics models, our analysis centers around distance distributions between different tags with the aim being to unravel the chromatin conformational arrangements. We show that for any specific genomic site, there are (at least) two different conformational arrangements of chromatin, implying coexisting distinct topologies which we refer to as phase α and phase β. These two phases show different scaling behaviors: the former is consistent with a crumpled globule, while the latter indicates a confined, but more extended conformation, such as a looped domain. The identification of these distinct phases sheds light on the coexistence of multiple chromatin topologies and provides insights into the effects of cellular context and/or treatments on chromatin structure.

To celebrate the 50th anniversary of Cell Press and the Cell focus issue on structural biology, we discussed with scientists working across diverse fields how AlphaFold has changed their research and brought structural biology to the masses., Competing Interests: Declaration of interests J.C.W. is a cofounder of MOMA Therapeutics, in which he has a financial interest. L.A.P., A.K.S., and H.E.X. are members of the Molecular Cell advisory board., (Copyright © 2023. Published by Elsevier Inc.)

Gene expression is the synthesis of proteins from the information encoded on DNA. One of the two main steps of gene expression is the translation of messenger RNA (mRNA) into polypeptide sequences of amino acids. Here, by taking into account mRNA degradation, we model the motion of ribosomes along mRNA with a ballistic model where particles advance along a filament without excluded volume interactions. Unidirectional models of transport have previously been used to fit the average density of ribosomes obtained by the experimental ribo-sequencing (Ribo-seq) technique in order to obtain the kinetic rates. The degradation rate is not, however, accounted for and experimental data from different experiments are needed to have enough parameters for the fit. Here, we propose an entirely novel experimental setup and theoretical framework consisting in splitting the mRNAs into categories depending on the number of ribosomes from one to four. We solve analytically the ballistic model for a fixed number of ribosomes per mRNA, study the different regimes of degradation, and propose a criterion for the quality of the inverse fit. The proposed method provides a high sensitivity to the mRNA degradation rate. The additional equations coming from using the monosome (single ribosome) and polysome (arbitrary number) ribo-seq profiles enable us to determine all the kinetic rates in terms of the experimentally accessible mRNA degradation rate., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chevalier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.

Tripartite motif-containing protein 21 (TRIM21) is a cytosolic antibody receptor and E3 ubiquitin ligase that promotes destruction of a broad range of pathogens. TRIM21 also underlies the antibody-dependent protein targeting method Trim-Away. Current evidence suggests that TRIM21 binding to antibodies leads to formation of a self-anchored K63 ubiquitin chain on the N terminus of TRIM21 that triggers the destruction of TRIM21, antibody, and target protein. Here, we report that addition of antibody and TRIM21 to Xenopus egg extracts promotes efficient degradation of endogenous target proteins, establishing cell-free Trim-Away as a powerful tool to interrogate protein function. Chemical methylation of TRIM21 had no effect on target proteolysis, whereas deletion of all lysine residues in targets abolished their ubiquitination and proteasomal degradation. These results demonstrate that target protein, but not TRIM21, polyubiquitination is required for Trim-Away, and they suggest that current models of TRIM21 function should be fundamentally revised., Competing Interests: Declaration of interests J.C.W. is a co-founder of MOMA therapeutics, in which he has a financial interest., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Endogenous and exogenous agents generate DNA-protein crosslinks (DPCs), whose replication-dependent degradation by the SPRTN protease suppresses aging and liver cancer. SPRTN is activated after the replicative CMG helicase bypasses a DPC and polymerase extends the nascent strand to the adduct. Here, we identify a role for the 5'-to-3' helicase FANCJ in DPC repair. In addition to supporting CMG bypass, FANCJ is essential for SPRTN activation. FANCJ binds ssDNA downstream of the DPC and uses its ATPase activity to unfold the protein adduct, which exposes the underlying DNA and enables cleavage of the adduct. FANCJ-dependent DPC unfolding is also essential for translesion DNA synthesis past DPCs that cannot be degraded. In summary, our results show that helicase-mediated protein unfolding enables multiple events in DPC repair., Competing Interests: Declaration of interests J.W. is a co-founder of MOMA Therapeutics, in which he has a financial interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The p97 ATPase extracts polyubiquitylated proteins from diverse cellular structures in preparation for destruction by the proteasome. p97 functions with Ufd1-Npl4 and a variety of UBA-UBX co-factors, but how p97 complexes assemble on ubiquitylated substrates is unclear. To address this, we investigated how p97 disassembles the CMG helicase after it is ubiquitylated during replication termination. We show that p97 Ufd1-Npl4 recruitment to CMG requires the UBA-UBX protein Ubxn7, and conversely, stable Ubxn7 binding to CMG requires p97 Ufd1-Npl4 . This cooperative assembly involves interactions between Ubxn7, p97, Ufd1-Npl4, and ubiquitin. Another p97 co-factor, Faf1, partially compensates for the loss of Ubxn7. Surprisingly, p97 Ufd1-Npl4-Ubxn7 and p97 Ufd1-Npl4-Faf1 also assemble cooperatively on unanchored ubiquitin chains. We propose that cooperative and substrate-independent recognition of ubiquitin chains allows p97 to recognize an unlimited number of polyubiquitylated proteins while avoiding the formation of partial, inactive complexes., (© 2022. The Author(s).)

The entry of mammalian cells into the DNA synthesis phase (S phase) represents a key event in cell division 1 . According to current models of the cell cycle, the kinase CDC7 constitutes an essential and rate-limiting trigger of DNA replication, acting together with the cyclin-dependent kinase CDK2. Here we show that CDC7 is dispensable for cell division of many different cell types, as determined using chemical genetic systems that enable acute shutdown of CDC7 in cultured cells and in live mice. We demonstrate that another cell cycle kinase, CDK1, is also active during G1/S transition both in cycling cells and in cells exiting quiescence. We show that CDC7 and CDK1 perform functionally redundant roles during G1/S transition, and at least one of these kinases must be present to allow S-phase entry. These observations revise our understanding of cell cycle progression by demonstrating that CDK1 physiologically regulates two distinct transitions during cell division cycle, whereas CDC7 has a redundant function in DNA replication., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The 5-hydroxymethylcytosine binding, embryonic stem-cell-specific (HMCES) protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in single-stranded DNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we use Xenopus egg extracts to show that an HMCES-DPC forms on the AP site generated during replication-coupled DNA interstrand cross-link repair. We show that HMCES cross-links form on DNA after the replicative CDC45-MCM2-7-GINS (CMG) helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses double-strand break formation, slows translesion synthesis past the AP site and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data demonstrate that HMCES-DPCs form as intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The bacterial flagellar motor is the membrane-embedded rotary motor, which turns the flagellum that provides thrust to many bacteria. This large multimeric complex, composed of a few dozen constituent proteins, is a hallmark of dynamic subunit exchange. The stator units are inner-membrane ion channels that dynamically bind to the peptidoglycan at the rotor periphery and apply torque. Their dynamic exchange is a function of the viscous load on the flagellum, allowing the bacterium to adapt to its local environment, although the molecular mechanisms of mechanosensitivity remain unknown. Here, by actively perturbing the steady-state stator stoichiometry of individual motors, we reveal a stoichiometry-dependent asymmetry in stator remodeling kinetics. We interrogate the potential effect of next-neighbor interactions and local stator unit depletion and find that neither can explain the observed asymmetry. We then simulate and fit two mechanistically diverse models that recapitulate the asymmetry, finding assembly dynamics to be particularly well described by a two-state catch-bond mechanism.

When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA interstrand cross-links (ICLs) covalently connect the two strands of the double helix and are extremely cytotoxic. Defective ICL repair causes the bone marrow failure and cancer predisposition syndrome, Fanconi anemia, and upregulation of repair causes chemotherapy resistance in cancer. The central event in ICL repair involves resolving the cross-link (unhooking). In this review, we discuss the chemical diversity of ICLs generated by exogenous and endogenous agents. We then describe how proliferating and nonproliferating vertebrate cells unhook ICLs. We emphasize fundamentally new unhooking strategies, dramatic progress in the structural analysis of the Fanconi anemia pathway, and insights into how cells govern the choice between different ICL repair pathways. Throughout, we highlight the many gaps that remain in our knowledge of these fascinating DNA repair pathways.

Cells employ transcription-coupled repair (TCR) to eliminate transcription-blocking DNA lesions. DNA damage-induced binding of the TCR-specific repair factor CSB to RNA polymerase II (RNAPII) triggers RNAPII ubiquitylation of a single lysine (K1268) by the CRL4 CSA ubiquitin ligase. How CRL4 CSA is specifically directed towards K1268 is unknown. Here, we identify ELOF1 as the missing link that facilitates RNAPII ubiquitylation, a key signal for the assembly of downstream repair factors. This function requires its constitutive interaction with RNAPII close to K1268, revealing ELOF1 as a specificity factor that binds and positions CRL4 CSA for optimal RNAPII ubiquitylation. Drug-genetic interaction screening also revealed a CSB-independent pathway in which ELOF1 prevents R-loops in active genes and protects cells against DNA replication stress. Our study offers key insights into the molecular mechanisms of TCR and provides a genetic framework of the interplay between transcriptional stress responses and DNA replication.

ParABS, the most widespread bacterial DNA segregation system, is composed of a centromeric sequence, parS, and two proteins, the ParA ATPase and the ParB DNA binding proteins. Hundreds of ParB proteins assemble dynamically to form nucleoprotein parS-anchored complexes that serve as substrates for ParA molecules to catalyze positioning and segregation events. The exact nature of this ParBS complex has remained elusive, what we address here by revisiting the Stochastic Binding model (SBM) introduced to explain the non-specific binding profile of ParB in the vicinity of parS. In the SBM, DNA loops stochastically bring loci inside a sharp cluster of ParB. However, previous SBM versions did not include the negative supercoiling of bacterial DNA, leading to use unphysically small DNA persistences to explain the ParB binding profiles. In addition, recent super-resolution microscopy experiments have revealed a ParB cluster that is significantly smaller than previous estimations and suggest that it results from a liquid-liquid like phase separation. Here, by simulating the folding of long (≥ 30 kb) supercoiled DNA molecules calibrated with realistic DNA parameters and by considering different possibilities for the physics of the ParB cluster assembly, we show that the SBM can quantitatively explain the ChIP-seq ParB binding profiles without any fitting parameter, aside from the supercoiling density of DNA, which, remarkably, is in accord with independent measurements. We also predict that ParB assembly results from a non-equilibrium, stationary balance between an influx of produced proteins and an outflux of excess proteins, i.e., ParB clusters behave like liquid-like protein condensates with unconventional "leaky" boundaries., Competing Interests: The authors have declared no competing interests exist.

DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or "nicks," which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, "runs off" the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates along double-stranded DNA beyond the break and is then ubiquitylated and removed from chromatin by the same pathway used during replication termination. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they suggest that CMG cannot be stored on dsDNA while cells resolve replication stress., Competing Interests: Declaration of interests J.C.W. is a co-founder of MoMa Therapeutics, in which he has a financial interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Translation is one of the main steps in the synthesis of proteins. It consists of ribosomes that translate sequences of nucleotides encoded on mRNA into polypeptide sequences of amino acids. Ribosomes bound to mRNA move unidirectionally, while unbound ribosomes diffuse in the cytoplasm. It has been hypothesized that finite diffusion of ribosomes plays an important role in ribosome recycling and that mRNA circularization enhances the efficiency of translation, see e.g. Lodish et al. (Molecular cell biology, 8th edn, W.H. Freeman and Company, San Francisco, 2016). In order to estimate the effect of cytoplasmic diffusion on the rate of translation, we consider a totally asymmetric simple exclusion process coupled to a finite diffusive reservoir, which we call the ribosome transport model with diffusion. In this model, we derive an analytical expression for the rate of protein synthesis as a function of the diffusion constant of ribosomes, which is corroborated with results from continuous-time Monte Carlo simulations. Using a wide range of biological relevant parameters, we conclude that diffusion is not a rate limiting factor in translation initiation because diffusion is fast enough in biological cells.

In preparation for cell division, the genome must be copied with high fidelity. However, replisomes often encounter obstacles, including bulky DNA lesions caused by reactive metabolites and chemotherapeutics, as well as stable nucleoprotein complexes. Here, we discuss recent advances in our understanding of TRAIP, a replisome-associated E3 ubiquitin ligase that is mutated in microcephalic primordial dwarfism. In interphase, TRAIP helps replisomes overcome DNA interstrand crosslinks and DNA-protein crosslinks, whereas in mitosis it triggers disassembly of all replisomes that remain on chromatin. We describe a model to explain how TRAIP performs these disparate functions and how they help maintain genome integrity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bacterial ParB partitioning proteins involved in chromosomes and low-copy-number plasmid segregation are cytosine triphosphate (CTP)-dependent molecular switches. CTP-binding converts ParB dimers to DNA clamps, allowing unidimensional diffusion along the DNA. This sliding property has been proposed to explain the ParB spreading over large distances from parS centromere sites where ParB is specifically loaded. We modeled such a "clamping and sliding" mechanism as a typical reaction-diffusion system, compared it to the F plasmid ParB DNA binding pattern, and found that it can account neither for the long range of ParB binding to DNA nor for the rapid assembly kinetics observed in vivo after parS duplication. Also, it predicts a strong effect on the F plasmid ParB binding pattern from the presence of a roadblock that is not observed in ChIP-sequencing (ChIP-seq). We conclude that although "clamping and sliding" can occur at short distances from parS , another mechanism must apply for ParB recruitment at larger genomic distances., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

When converging replication forks meet during replication termination, the CMG (Cdc45-MCM2-7-GINS) helicase is polyubiquitylated by CRL2 Lrr1 and unloaded from chromatin by the p97 ATPase. Here, we investigate the signal that triggers CMG unloading in Xenopus egg extracts using single-molecule and ensemble approaches. We show that converging CMGs pass each other and keep translocating at the same speed as before convergence, whereafter they are rapidly and independently unloaded. When CMG unloading is blocked, diverging CMGs do not support DNA synthesis, indicating that after bypass CMGs encounter the nascent lagging strands of the converging fork and then translocate along double-stranded DNA (dsDNA). However, translocation on dsDNA is not required for CMG's removal from chromatin because in the absence of nascent strand synthesis, converging CMGs are still unloaded. Moreover, recombinant CMG added to nuclear extract undergoes ubiquitylation and disassembly in the absence of any DNA, and DNA digestion triggers CMG ubiquitylation at stalled replication forks. Our findings suggest that DNA suppresses CMG ubiquitylation during elongation and that this suppression is relieved when CMGs converge, leading to CMG unloading., (© 2020 Low et al.; Published by Cold Spring Harbor Laboratory Press.)

Liquid-liquid phase-separated (LLPS) states are key to compartmentalizing components in the absence of membranes; however, it is unclear whether LLPS condensates are actively and specifically organized in the subcellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB), and a motor (ParA). We show that parS and ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favored by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates, and localizes non-canonical LLPS condensates in the subcellular space., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times., Competing Interests: Declaration of Interests J.E.C. is a co-founder of Spotlight Therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings identify a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair.

Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it uses DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using frog egg extracts that recapitulate NHEJ, we show that end processing requires the formation of a "short-range synaptic complex" in which DNA ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of end processing to a ligation-competent complex ensures that DNA ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Frog egg extracts represent a powerful approach with which to dissect molecular mechanisms of vertebrate DNA replication and repair. In the classical approach, sperm chromatin is added to a crude egg lysate to form replication-competent nuclei. We subsequently described a procedure that bypasses the requirement for nuclear assembly in DNA replication. In this method, DNA is first added to a high-speed supernatant (HSS) of egg lysate, which mimics the G 1 phase of the cell cycle in that it supports replication licensing. Subsequent addition of a concentrated nucleoplasmic extract (NPE) leads to replication initiation followed by a single complete round of DNA replication. The advantage of the nucleus-free system is that it supports efficient replication of model DNA templates such as plasmids and lambda DNA that can be modified with specific features such as LacI arrays, DNA protein cross-links, or DNA interstrand cross-links. Here, we describe our current protocol for preparation of HSS and NPE. Methods for their use in DNA replication and repair are described elsewhere., (© 2019 Cold Spring Harbor Laboratory Press.)

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink 1 ; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination 2 . It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings 3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chromosome and plasmid segregation in bacteria are mostly driven by ParAB S systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites ( parS ). However, the mechanism of how a few parS -bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico-mathematical models. We discriminated between these different models by varying some key parameters in vivo using the F plasmid partition system. We found that "Nucleation & caging" is the only coherent model recapitulating in vivo data. We also showed that the stochastic self-assembly of partition complexes (i) is a robust mechanism, (ii) does not directly involve ParA ATPase, (iii) results in a dynamic structure of discrete size independent of ParB concentration, and (iv) is not perturbed by active transcription but is by protein complexes. We refined the "Nucleation & caging" model and successfully applied it to the chromosomally encoded Par system of Vibrio cholerae , indicating that this stochastic self-assembly mechanism is widely conserved from plasmids to chromosomes., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Nonhomologous end joining (NHEJ) is the primary pathway of DNA double-strand-break repair in vertebrate cells, yet how NHEJ factors assemble a synaptic complex that bridges DNA ends remains unclear. To address the role of XRCC4-like factor (XLF) in synaptic-complex assembly, we used single-molecule fluorescence imaging in Xenopus laevis egg extract, a system that efficiently joins DNA ends. We found that a single XLF dimer binds DNA substrates just before the formation of a ligation-competent synaptic complex between DNA ends. The interaction of both globular head domains of the XLF dimer with XRCC4 is required for efficient formation of this synaptic complex. Our results indicate that, in contrast to a model in which filaments of XLF and XRCC4 bridge DNA ends, binding of a single XLF dimer facilitates the assembly of a stoichiometrically well-defined synaptic complex.

Background: The use of therapeutic antibodies for the treatment of neurological diseases is of increasing interest. Nose-to-brain drug delivery is one strategy to bypass the blood brain barrier. The neonatal Fc receptor (FcRn) plays an important role in transepithelial transcytosis of immunoglobulin G (IgG). Recently, the presence of the FcRn was observed in nasal respiratory mucosa. The aim of the present study was to determine the presence of functional FcRn in olfactory mucosa and to evaluate its role in drug delivery., Methods: Immunoreactivity and messenger RNA (mRNA) expression of FcRn was determined in ex vivo porcine olfactory mucosa. Uptake of IgG was performed in a side-by-side cell and analysed by immunofluorescence., Results: FcRn was found in epithelial and basal cells of the olfactory epithelium as well as in glands, cavernous bodies and blood vessels. Allogenic porcine IgGs were found time-dependently in the lamina propria and along axonal bundles, while only small amounts of xenogenic human IgGs were detected. Interestingly, lymphoid follicles were spared from allogenic IgGs., Conclusion: Fc-mediated transport of IgG across the nasal epithelial barrier may have significant potential for intranasal delivery, but the relevance of immune interaction in lymphoid follicles must be clarified to avoid immunogenicity., Competing Interests: left15364900© 2018 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

DNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.