DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5'-to-3' polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors

Mechanistic Insight Into Replication Fork Reversal Under Genotoxic Stress

Replication fork reversal - the transaction from a three-way junction (the usual conformation of replication forks) to a four-way junction - has been reported as a general response in face of a wide variety of genotoxic agents, oncogene activation and difficult-to-replicate regions of the genome. Proteins mediating this molecular transaction in vivo are still elusive. Many different groups of proteins have been proposed to execute this operation, yet most of the data come from biochemical experiments that may hardly recapitulate the complexity of this transaction in vivo. We focused our research on two groups of proteins, such as annealing helicases and post replicative repair (PRR) factors. The first group of proteins (SMARCAL1 and ZRANB3) have the unique ability to re-anneal RPA coated single stranded DNA (ssDNA), which may be necessary to convert standard replication forks to reversed forks. The second group of proteins (e.g. the E2 ubiquitin-conjugating enzyme UBC13 and polyubiquitinated PCNA) was selected as previous studies suggested the error-free branch of PRR to operate via template switching mechanisms, possibly entailing replication fork reversal. We could indeed determine that ZRANB3 is required for efficient fork slowing and reversal upon genotoxic stress and we are currently uncovering which domains are crucial for this function. We also uncovered the contribution of UBC13 and PCNA ubiquitination to active fork slowing and replication fork reversal upon genotoxic stress

Vitamin b12 metabolism in eukaryotes

Bibliography: p. 101Some pages are in colour

Development and Validation of 3D-CFD Injection and Combustion Models for Dual Fuel Combustion in Diesel Ignited Large Gas Engines

This paper focuses on improving the 3D-Computational Fluid Dynamics (CFD) modeling of diesel ignited gas engines, with an emphasis on injection and combustion modeling. The challenges of modeling are stated and possible solutions are provided. A specific approach for modeling injection is proposed that improves the modeling of the ballistic region of the needle lift. Experimental results from an inert spray chamber are used for model validation. Two-stage ignition methods are described along with improvements in ignition delay modeling of the diesel ignited gas engine. The improved models are used in the Extended Coherent Flame Model with the 3 Zones approach (ECFM-3Z). The predictive capability of the models is investigated using data from single cylinder engine (SCE) tests conducted at the Large Engines Competence Center (LEC). The results are discussed and further steps for development are identified

Deep learning is widely applicable to phenotyping embryonic development and disease

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview

Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy

Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition

Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal through a process that requires poly(ADP-ribose) polymerase (PARP) activity. Whether regressed forks can efficiently restart and what factors are required to restart fork progression after fork reversal are still unknown. We have combined biochemical and EM approaches with single-molecule DNA fiber analysis to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition that is not shared by other human RecQ proteins. We show that the poly(ADP-ribosyl)ation activity of PARP1 stabilizes forks in the regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition