ZNF827 is a single-stranded DNA binding protein that regulates the ATR-CHK1 DNA damage response pathway.

The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.

Nuclear export of circular RNA.

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.

Fancm has dual roles in the limiting of meiotic crossovers and germ cell maintenance in mammals.

Meiotic crossovers are required for accurate chromosome segregation and producing new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we reveal the tumor suppressor FANCM as a meiotic anti-crossover factor in mammals. We use unique large-scale crossover analyses with both single-gamete sequencing and pedigree-based bulk-sequencing datasets to identify a genome-wide increase in crossover frequencies in Fancm-deficient mice. Gametogenesis is heavily perturbed in Fancm loss-of-function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway after birth. Despite the gametogenesis phenotypes in Fancm mutants, both sexes are capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders.

Branchpoint translocation by fork remodelers as a general mechanism of R-loop removal.

Co-transcriptional R loops arise from stalling of RNA polymerase, leading to the formation of stable DNA:RNA hybrids. Unresolved R loops promote genome instability but are counteracted by helicases and nucleases. Here, we show that branchpoint translocases are a third class of R-loop-displacing enzyme in vitro. In cells, deficiency in the Fanconi-anemia-associated branchpoint translocase FANCM causes R-loop accumulation, particularly after treatment with DNA:RNA-hybrid-stabilizing agents. This correlates with FANCM localization at R-loop-prone regions of the genome. Moreover, other branchpoint translocases associated with human disease, such as SMARCAL1 and ZRANB3, and those from lower organisms can also remove R loops in vitro. Branchpoint translocases are more potent than helicases in resolving R loops, indicating their evolutionary important role in R-loop suppression. In human cells, FANCM, SMARCAL1, and ZRANB3 depletion causes additive effects on R-loop accumulation and DNA damage. Our work reveals a mechanistic basis for R-loop displacement that is linked to genome stability.

Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability.

The RecQ-like helicase BLM cooperates with topoisomerase IIIα, RMI1, and RMI2 in a heterotetrameric complex (the "Bloom syndrome complex") for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIα are coupled, we purified the active four-subunit complex. Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.

Cyclin-Dependent Kinase-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity.

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs upon exposure to DNA-damaging agents and during the S phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a cyclin-dependent kinase (CDK) regulatory phosphosite (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by immunoblotting with an S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during the S phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity.

The Fanconi anemia ubiquitin E3 ligase complex as an anti-cancer target.

Agents that induce DNA damage can cure some cancers. However, the side effects of chemotherapy are severe because of the indiscriminate action of DNA-damaging agents on both healthy and cancerous cells. DNA repair pathway inhibition provides a less toxic and targeted alternative to chemotherapy. A compelling DNA repair target is the Fanconi anemia (FA) E3 ligase core complex due to its critical-and likely singular-role in the efficient removal of specific DNA lesions. FA pathway inactivation has been demonstrated to specifically kill some types of cancer cells without the addition of exogenous DNA damage, including cells that lack BRCA1, BRCA2, ATM, or functionally related genes. In this perspective, we discuss the genetic and biochemical evidence in support of the FA core complex as a compelling drug target for cancer therapy. In particular, we discuss the genetic, biochemical, and structural data that could rapidly advance our capacity to identify and implement the use of FA core complex inhibitors in the clinic.

The ubiquitination machinery of the Fanconi Anemia DNA repair pathway.

The Fanconi Anemia (FA) pathway maintains genome stability by preventing DNA damage from occurring when replication is blocked. Central to the FA pathway is the monoubiquitination of FANCI-FANCD2 mediated by a ubiquitin RING-E3 ligase complex called the FA core complex. Genetic mutation in any component of the FA core complex results in defective FANCI-FANCD2 monoubiquitination and phenotypes of DNA damage sensitivity, birth defects, early-onset bone marrow failure and cancer. Here, we discuss the mechanisms of the FA core complex and FANCI-FANCD2 monoubiquitination at sites of blocked replication and review our current understanding of the biological functions of these proteins in replication fork protection.

A Structural Guide to the Bloom Syndrome Complex.

The Bloom syndrome complex is a DNA damage repair machine. It consists of several protein components which are functional in isolation, but interdependent in cells for the maintenance of accurate homologous recombination. Mutations to any of the genes encoding these proteins cause numerous physical and developmental markers as well as phenotypes of genome instability, infertility, and cancer predisposition. Here we review the published structural and biochemical data on each of the components of the complex: the helicase BLM, the type IA topoisomerase TOP3A, and the OB-fold-containing RMI and RPA subunits. We describe how each component contributes to function, interacts with each other, and the DNA that it manipulates/repairs.

Formaldehyde Causes Bone Marrow Failure Linked to Transcriptional Reprogramming or Metabolic Deficiency.

Two new studies in this issue of Molecular Cell demonstrate that bone marrow failure, in mice and humans, can be induced by formaldehyde generated either from defective metabolism (Dingler et al., 2020) or during the process of transcriptional reprogramming (Shen et al., 2020).

Structural insight into FANCI-FANCD2 monoubiquitination.

The Fanconi anemia (FA) pathway coordinates a faithful repair mechanism for DNA damage that blocks DNA replication, such as interstrand cross-links. A key step in the FA pathway is the conjugation of ubiquitin on to FANCD2 and FANCI, which is facilitated by a large E3 ubiquitin ligase complex called the FA core complex. Mutations in FANCD2, FANCI or FA core complex components cause the FA bone marrow failure syndrome. Despite the importance of these proteins to DNA repair and human disease, our molecular understanding of the FA pathway has been limited due to a deficit in structural studies. With the recent development in cryo-electron microscopy (EM), significant advances have been made in structural characterization of these proteins in the last 6 months. These structures, combined with new biochemical studies, now provide a more detailed understanding of how FANCD2 and FANCI are monoubiquitinated and how DNA repair may occur. In this review, we summarize these recent advances in the structural and molecular understanding of these key components in the FA pathway, compare the activation steps of FANCD2 and FANCI monoubiquitination and suggest molecular steps that are likely to be involved in regulating its activity.

Methodology for the identification of small molecule inhibitors of the Fanconi Anaemia ubiquitin E3 ligase complex.

DNA inter-strand crosslinks (ICLs) threaten genomic stability by creating a physical barrier to DNA replication and transcription. ICLs can be caused by endogenous reactive metabolites or from chemotherapeutics. ICL repair in humans depends heavily on the Fanconi Anaemia (FA) pathway. A key signalling step of the FA pathway is the mono-ubiquitination of Fanconi Anaemia Complementation Group D2 (FANCD2), which is achieved by the multi-subunit E3 ligase complex. FANCD2 mono-ubiquitination leads to the recruitment of DNA repair proteins to the site of the ICL. The loss of FANCD2 mono-ubiquitination is a common clinical feature of FA patient cells. Therefore, molecules that restore FANCD2 mono-ubiquitination could lead to a potential drug for the management of FA. On the other hand, in some cancers, FANCD2 mono-ubiquitination has been shown to be essential for cell survival. Therefore, inhibition of FANCD2 mono-ubiquitination represents a possible therapeutic strategy for cancer specific killing. We transferred an 11-protein FANCD2 mono-ubiquitination assay to a high-throughput format. We screened 9,067 compounds for both activation and inhibition of the E3 ligase complex. The use of orthogonal assays revealed that candidate compounds acted via non-specific mechanisms. However, our high-throughput biochemical assays demonstrate the feasibility of using sophisticated and robust biochemistry to screen for small molecules that modulate a key step in the FA pathway. The future identification of FA pathway modulators is anticipated to guide future medicinal chemistry projects with drug leads for human disease.

CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.

ATR-Mediated FANCI Phosphorylation Regulates Both Ubiquitination and Deubiquitination of FANCD2.

DNA interstrand crosslinks (ICLs) are a physical barrier to replication and therefore toxic to cell viability. An important mechanism for the removal of ICLs is the Fanconi Anemia DNA repair pathway, which is initiated by mono-ubiquitination of FANCD2 and its partner protein FANCI. Here, we show that maintenance of FANCD2 and FANCI proteins in a monoubiquitinated form is regulated by the ATR-kinase. Using recombinant proteins in biochemical reconstitution experiments we show that ATR directly phosphorylates FANCI on serine 556, 559, and 565 to stabilize its association with DNA and FANCD2. This increased association with DNA stimulates the conjugation of ubiquitin to both FANCI and FANCD2, but also inhibits ubiquitin deconjugation. Using phosphomimetic and phosphodead mutants of FANCI we show that S559 and S565 are particularly important for protecting the complex from the activity of the deubiquitinating enzyme USP1:UAF1. Our results reveal a major mechanism by which ATR kinase maintains the activation of the FA pathway, by promoting the accumulation of FANCD2 in the ubiquitinated form active in DNA repair.

Monoubiquitination by the human Fanconi anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays.

FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show using electron microscopy that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.

Bone marrow is the spongy tissue inside bones that produces blood cells. Fanconi anemia is the most common form of inherited bone marrow death and affects children and young adults. In this disease, bone marrow cells cannot attach a protein tag called ubiquitin to another protein called FANCD2. When DNA becomes damaged, FANCD2 helps cells to respond and repair the damage but without ubiquitin it cannot do this correctly. Without ubiquitin linked to FANCD2 bone marrow cells die from damaged DNA. Another protein, called FANCI, works in partnership with FANCD2 and also gets linked to ubiquitin. Tan et al. studied purified proteins in the laboratory to understand how linking ubiquitin changes the behavior of FANCD2 and FANCI. When the proteins have ubiquitin attached, they can form stable attachments to DNA. Without ubiquitin, however, the proteins only attach to DNA for short periods of time. Using electron microscopy, Tan et al. discovered that large numbers of the modified proteins become tightly attached to damaged DNA, helping to protect it and triggering DNA repair processes. Understanding the role of FANCD2 in Fanconi anemia could lead to new treatments. FANCD2 and FANCI have similar roles in other cells too. Stopping them from protecting damaged DNA in cancer cells could be used to enhance the success of chemotherapies and radiotherapies.

Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin.

Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.