Interstrand crosslink

The Fanconi anemia (FA) pathway is a key pathway involved in the repair of DNA interstrand crosslinking (ICL) damage, which chiefly includes the following four modules: lesion recognition, FA core complex recruitment, FANCD2-FANCI complex monoubiquitination, and downstream events (nucleolytic incision, translesion synthesis, and homologous recombination). Mutations or deletions of multiple FA genes in this pathway can damage the ICL repair pathway and disrupt primordial germ cell development and oocyte meiosis, thereby leading to abnormal follicular development...

Saccharomyces cerevisiae Pso2/SNM1 is essential for DNA interstrand crosslink (ICL) repair; however, its mechanism of action remains incompletely understood. While recent work has revealed that Pso2/Snm1 is dual-localized in the nucleus and mitochondria, it remains unclear whether cell-intrinsic and -extrinsic factors regulate its subcellular localization and function. Herein, we show that Pso2 undergoes ubiquitination and phosphorylation, but not SUMOylation, in unstressed cells. Unexpectedly, we found that methyl methanesulfonate (MMS), rather than ICL-forming agents, induced robust SUMOylation of Pso2 on two conserved residues, K97 and K575, and that SUMOylation markedly increased its abundance in the mitochondria...

FAAP20 is a Fanconi anemia (FA) protein that associates with the FA core complex to promote FANCD2/FANCI monoubiquitination and activate the damage response to interstrand crosslink damage. Here, we report that FAAP20 has a marked role in homologous recombination at a DNA double-strand break not associated with an ICL and separable from its binding partner FANCA. While FAAP20's role in homologous recombination is not dependent on FANCA, we found that FAAP20 stimulates FANCA's biochemical activity in vitro and participates in the single-strand annealing pathway of double-strand break repair in a FANCA-dependent manner...

While natural oligonucleotides (ONs) are increasingly used as therapeutic and diagnostic tools, they still face certain challenges such as low resistance to enzymatic degradation, potential immunogenicity, and delivery issues, which can limit their applications. Peptide Nucleic Acids (PNAs) are promising alternatives due to their high affinity for DNA and RNA, the high resistance to enzymatic degradation, and the easy introduction of a wide range of potential modifications. Chemical modifications that enable the covalent targeting of specific DNA and RNA strands offer additional advantages, including enhanced potency...

Oligonucleotides (ODNs) find applications as diagnostic and therapeutic tools due to their unique ability to interact, thanks to Watson-Crick base pairing, with a specific DNA or RNA target strand. Although most of the tools available today rely on mere hydrogen bond formation, chemical modifications to enable covalent interstrand-crosslinking (ICL) have been reported, and are gaining a place under the spotlight as they potentially offer a series of advantages over the state of the art, including a higher potency and selectivity...

Purpose: Ionizing radiation (IR)-induced transcriptional changes are considered a potential biodosimetry for dose evaluation and health risk monitoring of acute or chronic radiation exposure. It is crucial to understand the impact of confounding factors on the radiation-responsive gene expressions for accurate and reproducible dose assessment. This study aims to explore the potential influence of exposures to chemotherapeutic agents such as cyclophosphamide (CP) and mitomycin C (MMC) on IR-induced the transcriptional biomarkers...

FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea-induced stalled forks. The RAD51 recombinase has also been implicated in regulation of resection at stalled replication forks. The mechanistic contributions of these proteins to fork protection are not well understood. Here, we used purified FANCD2 and RAD51 to study how each protein regulates DNA resection at stalled forks. We characterized three mechanisms of FANCD2-mediated fork protection: (1) The N-terminal domain of FANCD2 inhibits the essential DNA2 nuclease activity by directly binding to DNA2 accounting for over-resection in FANCD2 defective cells...

Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a Fancg -KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire Fancg locus was targeted and maintained on the immunological well-characterized C57BL/6J background...

Fanconi anemia (FA) is a genetic disorder associated with developmental defects, bone marrow failure and cancer. The FA pathway is crucial for the repair of DNA interstrand crosslinks (ICLs). In this study, we have developed and characterized a new tool to investigate ICL repair: a clickable version of the crosslinking agent melphalan which we name click-melphalan. Our results demonstrate that click-melphalan is as effective as its unmodified counterpart in generating ICLs and associated toxicity. The lesions induced by click-melphalan can be detected in cells by post-labelling with a fluorescent reporter and quantified using flow cytometry...

The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs) in humans. Activation of the pathway relies on loading of the FANCD2/FANCI complex onto chromosomes, where it is fully activated by subsequent monoubiquitination. However, the mechanism for loading the complex onto chromosomes remains unclear. Here, we identify 10 SQ/TQ phosphorylation sites on FANCD2, which are phosphorylated by ATR in response to ICLs. Using a range of biochemical assays complemented with live-cell imaging including super-resolution single-molecule tracking, we show that these phosphorylation events are critical for loading of the complex onto chromosomes and for its subsequent monoubiquitination...

Immunofluorescence imaging is a standard experimental tool for monitoring the response of cellular factors to DNA damage. Visualizing the recruitment of DNA Damage Response (DDR) components requires high affinity antibodies, which are generally available. In contrast, reagents for the display of the lesions that induce the response are far more limited. Consequently, DDR factor accumulation often serves as a surrogate for damage, without reporting the actual inducing structure. This limitation has practical implications given the importance of the response to DNA reactive drugs such as those used in cancer therapy...

The replication machinery frequently encounters DNA damage and other structural impediments that inhibit progression of the replication fork. Replication-coupled processes that remove or bypass the barrier and restart stalled forks are essential for completion of replication and for maintenance of genome stability. Errors in replication-repair pathways lead to mutations and aberrant genetic rearrangements and are associated with human diseases. This review highlights recent structures of enzymes involved in three replication-repair pathways: translesion synthesis, template switching and fork reversal, and interstrand crosslink repair...

DNA interstrand crosslinks (ICLs) pose a major obstacle for DNA replication and transcription if left unrepaired. The cellular response to ICLs requires the coordination of various DNA repair mechanisms. Homologous recombination (HR) intermediates generated in response to ICLs, require efficient and timely conversion by structure-selective endonucleases. Our knowledge on the precise coordination of this process remains incomplete. Here, we designed complementary genetic screens to map the machinery involved in the response to ICLs and identified FIRRM/C1orf112 as an indispensable factor in maintaining genome stability...

BACKGROUND: Renal medullary carcinoma (RMC) is a highly aggressive cancer in need of new therapeutic strategies. The neddylation pathway can protect cells from DNA damage induced by the platinum-based chemotherapy used in RMC. We investigated if neddylation inhibition with pevonedistat will synergistically enhance antitumour effects of platinum-based chemotherapy in RMC. METHODS: We evaluated the IC50 concentrations of the neddylation-activating enzyme inhibitor pevonedistat in vitro in RMC cell lines...

UNLABELLED: DNA interstrand crosslinks (ICLs) are covalent bonds between opposing strands of the DNA helix which prevent DNA melting and subsequent DNA replication or RNA transcription. Here, we show that UV Stimulated Scaffold Protein A (UVSSA) participates in transcription-coupled repair of ICLs. UVSSA encodes a protein that regulates the activity of RNA polymerase II (Pol II) to facilitate the repair of UV lesions and to relieve transcription stress resulting from MYC activation. We show that UVSSA knockout sensitizes cells to ICL-inducing drugs, that UVSSA is specifically required for transcription-coupled repair of a single ICL in a fluorescence-based reporter assay, and that transcription-coupled repair of ICLs is defective in UVSSA -/- cells...

Fanconi anemia (FA) is a rare inherited disorder that mainly affects the bone marrow. This condition causes decreased production of all types of blood cells. FA is caused by a defective repair of DNA interstrand crosslinks and to date, mutations in over 20 genes have been linked to the disease. Advances in science and molecular biology have provided new insight between FA gene mutations and the severity of clinical manifestations. Here, we will highlight the current and promising therapeutic options for this rare disease...

Activation of the Fanconi anemia (FA) pathway after treatment with mitomycin C (MMC) is essential for preventing chromosome translocations termed "radials." When replication forks stall at MMC-induced interstrand crosslinks (ICLs), the FA pathway is activated to orchestrate ICL unhooking and repair of the DNA break intermediates. However, in FA-deficient cells, how ICL-associated breaks are resolved in a manner that leads to radials is unclear. Here, we demonstrate that MMC-induced radials are dependent on DNA polymerase theta (POLθ)-mediated alternative end joining (A-EJ)...

Covalent crosslinking of DNA strands provides a useful tool for medical, biochemical, and DNA nanotechnology applications. Here we present a light-induced interstrand DNA crosslinking reaction using the modified nucleoside 5-phenylethynyl-2'-deoxyuridine (Phe dU). The crosslinking ability of Phe dU was programmed by base pairing and by metal ion interaction at the Watson-Crick base pairing site. Rotation to intrahelical positions was favored by hydrophobic stacking and enabled an unexpected photochemical alkene-alkyne [2 + 2] cycloaddition within the DNA duplex, resulting in efficient formation of a Phe dU dimer after short irradiation times of a few seconds...

Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital abnormalities and increased risk for bone marrow failure and cancer. FA is caused by mutation of any one of 23 genes, the protein products of which function primarily in the maintenance of genome stability. An important role for the FA proteins in the repair of DNA interstrand crosslinks (ICLs) has been established in vitro . While the endogenous sources of ICLs relevant to the pathophysiology of FA have yet to be clearly determined, a role for the FA proteins in a two-tier system for the detoxification of reactive metabolic aldehydes has been established...

SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway...