Role In Interstrand Crosslink Repair Research Articles

The RPA inhibitor HAMNO sensitizes Fanconi anemia pathway-deficient cells

ABSTRACT The Fanconi anemia (FA) DNA repair pathway is required for DNA inter-strand crosslink (ICL) repair. Besides its role in ICL repair, FA proteins play a central role in stabilizing stalled replication forks, thereby ensuring genome integrity. We previously demonstrated that depletion of replication protein A (RPA) induces the activation of FA pathway leading to FANCD2 monoubiquitination and FANCD2 foci formation. Thus, we speculated that FA-deficient cells would be more sensitive to RPA inhibition compared to FA-proficient cells. Following treatment with RPA inhibitor HAMNO, we observed significant induction in FANCD2 monoubiquitination and foci formation as observed in RPA depletion. In addition, HAMNO treatment caused increased levels of γ-H2AX and S-phase accumulation in FA-deficient cells. Importantly, FA-deficient cells showed more increased sensitivity to HAMNO than FA-proficient cells. Moreover, in combination with cisplatin, HAMNO further enhanced the cytotoxicity of cisplatin in FA-deficient cells, while being less toxic against FA-proficient cells. This result suggests that RPA inhibition might be a potential therapeutic candidate for the treatment of FA pathway-deficient tumors.

Lnk/Sh2b3 Deficiency Restores Hematopoietic Stem Cell Function and Genome Integrity in Fanconi Anemia

▪Although initially identified over 85 years ago, Fanconi Anemia (FA) remains a fatal genetic disease. Patients with FA exhibit developmental malformations, bone marrow failure, and increased cancer susceptibility. Twenty-one FA genes cooperate in a genome stability pathway that is essential for repair of DNA interstrand crosslinks (ICLs) and attenuation of replication stress. Cells derived from FA patients are hypersensitive to ICL-inducing agents such as Mitomycin C (MMC), a hallmark of FA. In addition, FA hematopoietic stem and progenitor cells (HSPCs) show growth retardation and increased apoptosis culminating in hematopoietic stem cell (HSC) exhaustion and bone marrow failure. The poor growth of HSPCs also poses a significant obstacle in gene therapy for FA patients. Moreover, unrepaired DNA damage increases genome instability and promotes leukemia and cancer. Of note, interventions to mitigate HSPC defects in FA do not exist, aside from allogeneic bone marrow transplantation.Remarkably, we reveal here that loss of the adaptor protein Lnk (also called Sh2b3) suppresses FA associated phenotypes without further accelerating neoplastic transformation. LNK is a negative regulator of HSC self-renewal in part by attenuating cytokine signaling, in particular thrombopoietin (TPO)-activated JAK2 signaling in HSCs. Fancd2-/-;Lnk-/- mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals. The reconstituting ability of Fancd2-/-;Lnk-/- HSCs is also largely restored to WT levels in serial transplantation assays. In addition, Lnk deficiency restored the growth and survival of FA HSPCs in ex vivo culture. More importantly, Lnk deficiency mitigated chromosomal aberrations associated with FA upon proliferation. To determine if Lnk deficiency mitigates ICL repair, we examined MMC sensitivity in primary HSPCs that were dually null for Lnk and FancD2. Our data suggest that LNK does not play an overt role in ICL repair. Rather, Lnk deficiency in HSPCs mitigated replication stress by stabilizing replication forks and reduces genome instability associated with FA. Hence, our data suggest that Lnk deficiency alleviates replication stress-induced DNA damage and genome instability, thereby restoring cell proliferation and survival of Fancd2-/- HSPCs ex vivo and HSC functions in vivo.Last and importantly, we began to test if LNK inhibition restores human FA HSPCs. We first generated FA-like HSPCs by knocking down FANCD2 in normal umbilical cord blood (UCB) CD34+ cells. Our results demonstrated that shRNA-mediated depletion of LNK in human CD34+ cells rescued progenitor cell numbers and growth in FA-like HSPCs. Take together, our findings reveal a previously unappreciated role of cytokine/JAK signaling in alleviating replication stress and promoting replication-associated genome maintenance. Our studies also illuminate replication stress as a major underlying origin of bone marrow failure in FA patients and have implications for therapeutic strategies to treat FA associated bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Structural mechanism of DNA interstrand cross-link unhooking by the bacterial FAN1 nuclease

DNA interstrand cross-links (ICLs) block the progress of the replication and transcription machineries and can weaken chromosomal stability, resulting in various diseases. FANCD2-FANCI-associated nuclease (FAN1) is a conserved structure-specific nuclease that unhooks DNA ICLs independently of the Fanconi anemia pathway. Recent structural studies have proposed two different mechanistic features for ICL unhooking by human FAN1: a specific basic pocket that recognizes the terminal phosphate of a 1-nucleotide (nt) 5' flap or FAN1 dimerization. Herein, we show that despite lacking these features, Pseudomonas aeruginosa FAN1 (PaFAN1) cleaves substrates at ∼3-nt intervals and resolves ICLs. Crystal structures of PaFAN1 bound to various DNA substrates revealed that its conserved basic Arg/Lys patch comprising Arg-228 and Lys-260 recognizes phosphate groups near the 5' terminus of a DNA substrate with a 1-nt flap or a nick. Substitution of Lys-260 did not affect PaFAN1's initial endonuclease activity but significantly decreased its subsequent exonuclease activity and ICL unhooking. The Arg/Lys patch also interacted with phosphates at a 3-nt gap, and this interaction could drive movement of the scissile phosphates into the PaFAN1-active site. In human FAN1, the ICL-resolving activity was not affected by individual disruption of the Arg/Lys patch or basic pocket. However, simultaneous substitution of both FAN1 regions significantly reduced its ICL-resolving activity, suggesting that these two basic regions play a complementary role in ICL repair. On the basis of these findings, we propose a conserved role for two basic regions in FAN1 to guide ICL unhooking and to maintain genomic stability.

Systematic analysis of DNA crosslink repair pathways during development and aging in Caenorhabditis elegans.

DNA interstrand crosslinks (ICLs) are generated by endogenous sources and chemotherapeutics, and pose a threat to genome stability and cell survival. Using Caenorhabditis elegans mutants, we identify DNA repair factors that protect against the genotoxicity of ICLs generated by trioxsalen/ultraviolet A (TMP/UVA) during development and aging. Mutations in nucleotide excision repair (NER) components (e.g. XPA-1 and XPF-1) imparted extreme sensitivity to TMP/UVA relative to wild-type animals, manifested as developmental arrest, defects in adult tissue morphology and functionality, and shortened lifespan. Compensatory roles for global-genome (XPC-1) and transcription-coupled (CSB-1) NER in ICL sensing were exposed. The analysis also revealed contributions of homologous recombination (BRC-1/BRCA1), the MUS-81, EXO-1, SLX-1 and FAN-1 nucleases, and the DOG-1 (FANCJ) helicase in ICL resolution, influenced by the replicative-status of the cell/tissue. No obvious or critical role in ICL repair was seen for non-homologous end-joining (cku-80) or base excision repair (nth-1, exo-3), the Fanconi-related proteins BRC-2 (BRCA2/FANCD1) and FCD-2 (FANCD2), the WRN-1 or HIM-6 (BLM) helicases, or the GEN-1 or MRT-1 (SNM1) nucleases. Our efforts uncover replication-dependent and -independent ICL repair networks, and establish nematodes as a model for investigating the repair and consequences of DNA crosslinks in metazoan development and in adult post-mitotic and proliferative germ cells.

LNK (SH2B3) Deficiency Ameliorates Hematopoietic Stem Cell Defects in Fanconi Anemi

Fanconi Anemia (FA) is one of the most common inherited bone marrow failure (iBMF) syndromes. Although initially identified over 85 years ago, FA remains a fatal genetic disease. Nineteen FA genes cooperate in a genome stability pathway that is essential for repair of DNA damage and tolerance of replication stress. Cells derived from FA patients are hypersensitive to DNA interstrand crosslink (ICL)-inducing agents, such as Mitomycin C (MMC), and exhibit DNA damage checkpoint and mitosis defects. Mutations in FA genes result in hematopoietic stem cell (HSC) defects, bone marrow failure and cancer predisposition. Importantly, interventions to mitigate HSC defects do not exist, aside from allogeneic bone marrow transplantation (BMT).HSCs deficient for FancD2, a central component of the FA signaling pathway, are markedly compromised in reconstituting the hematopoietic system in murine BMT models. Remarkably, we found that loss of the adaptor protein Lnk (also called Sh2b3) restores HSC function in FancD2 knockout mice without accelerating neoplastic transformation. LNK negatively regulates HSC self-renewal, in part by attenuating cytokine signaling-activated JAK2 signaling in HSCs (J Clin Invest. 2008;118(8):2832-2844). Fancd2-/-;Lnk-/- (DKO) mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals, and DKOHSC function is also largely restored to WT levels in serial transplantation assays. Primary DKOHSC and progenitors (HSPCs) are still sensitive to MMC, indicating that LNK does not play an overt role in ICL repair. Instead, Lnk deficiency notably reduces spontaneous DNA damage and genome instability. This is in agreement with recent studies that reveal a requirement for FA proteins in replication stress, which is a separable function from their role in DNA repair (Cell. 2011;145(4):529-542; Cancer Cell. 2012;22(1):106-116). Strikingly, we demonstrated that Lnk deficiency mitigates replication stress by stabilizing stalled replication forks, and that this effect is dependent upon cytokine signaling.Together, our data demonstrate that Lnk deficiency ameliorates FA HSPC defects by alleviating replication stress associated DNA damage and genome instability. To our knowledge, this is one of the first examples of in vivo genetic suppression of FA-associated HSPC defects. Our work sheds light on mechanisms underlying the origin of bone marrow failure in FA patients and has implications for new therapeutic strategies to treat FA associated bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a Kd for DNA of 9.2 nm, with an off-rate constant of 0.013 s(-1)and an on-rate constant of 14 μm(-1) s(-1). dNTP binding is rapid with Kd values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a Kd value for PPi of 3.7 mm and a maximal rate constant of 11 s(-1). Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s(-1); rapid phosphodiester bond formation occurs with a Keq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s(-1), for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.

Hrq1, a Homolog of the Human RecQ4 Helicase, Acts Catalytically and Structurally to Promote Genome Integrity

Human RecQ4 (hRecQ4) affects cancer and aging but is difficult to study because it is a fusion between a helicase and an essential replication factor. Budding yeast Hrq1 is homologous to the disease-linked helicase domain of RecQ4 and, like hRecQ4, is a robust 3'-5' helicase. Additionally, Hrq1 has the unusual property of forming heptameric rings. Cells lacking Hrq1 exhibited two DNA damage phenotypes: hypersensitivity to DNA interstrand crosslinks (ICLs) and telomere addition to DNA breaks. Both activities are rare; their coexistence in a single protein is unprecedented. Resistance to ICLs requires helicase activity, but suppression of telomere addition does not. Hrq1 also affects telomere length by a noncatalytic mechanism, as well as telomerase-independent telomere maintenance. Because Hrq1 binds telomeres in vivo, it probably affects them directly. Thus, the tumor-suppressing activity of RecQ4 could be due to a role in ICL repair and/or suppression of de novo telomere addition.

Coordinate Nuclear Targeting of the FANCD2 and FANCI Proteins via a FANCD2 Nuclear Localization Signal

Fanconi anemia (FA) is a rare recessive disease, characterized by congenital defects, bone marrow failure, and increased cancer susceptibility. FA is caused by biallelic mutation of any one of sixteen genes. The protein products of these genes function cooperatively in the FA-BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of this pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to discrete chromatin regions where they function in ICL repair. Despite their critical role in ICL repair, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, or how they are targeted to the nucleus and chromatin. In this study, we describe the functional characterization of an amino-terminal FANCD2 nuclear localization signal (NLS). We demonstrate that the amino terminal 58 amino acids of FANCD2 can promote the nuclear expression of GFP and is necessary for the nuclear localization of FANCD2. Importantly, mutation of this FANCD2 NLS reveals that intact FANCD2 is required for the nuclear localization of a subset of FANCI. In addition, the NLS is necessary for the efficient monoubiquitination of FANCD2 and FANCI and, consequently, for their localization to chromatin. As a result, FANCD2 NLS mutants fail to rescue the ICL sensitivity of FA-D2 patient cells. Our studies yield important insight into the domain structure of the poorly characterized FANCD2 protein, and reveal a previously unknown mechanism for the coordinate nuclear import of a subset of FANCD2 and FANCI, a key early step in the cellular ICL response.

Snm1B Interacts with PSF2

The protein Snm1B plays a key role in interstrand crosslink (ICL) repair. In a yeast two-hybrid screen we identified the protein PSF2 to bind Snm1B. PSF2 is a member of the GINS complex involved in replication initiation and elongation, and is known to play a role in ICL repair. Snm1B was shown to bind PSF2 in human cells through two regions, strongly to a 144 amino acid N-terminal region and weakly to a second smaller 37 amino acid C-terminal region. Ectopic expression of PSF2 increased the amount of Mus81, a protein component of the endonucleolytic complex involved in ICL repair, co-immunoprecipitating with Snm1B. Moreover, deleting the N-terminal, but not C-terminal region of Snm1B reduced the amount of co-immunoprecipitated Mus81. Conversely, the telomere-binding protein TRF2 competed with PSF2 for binding to the C-terminus of Snm1B, and deletion of this region, but not the N-terminal region, reduced Snm1B chromatin association. We speculate that the N-terminal region of Snm1B forms a complex containing PSF2 and Mus81, while the C-terminal region is important for PSF2-mediated chromatin association.

The phenotype of FancB-mutant mouse embryonic stem cells

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

Drosophila MUS312 and the Vertebrate Ortholog BTBD12 Interact with DNA Structure-Specific Endonucleases in DNA Repair and Recombination

DNA recombination and repair pathways require structure-specific endonucleases to process DNA structures that include forks, flaps, and Holliday junctions. Previously, we determined that the Drosophila MEI-9-ERCC1 endonuclease interacts with the MUS312 protein to produce meiotic crossovers, and that MUS312 has a MEI-9-independent role in interstrand crosslink (ICL) repair. The importance of MUS312 to pathways crucial for maintaining genomic stability in Drosophila prompted us to search for orthologs in other organisms. Based on sequence, expression pattern, conserved protein-protein interactions, and ICL repair function, we determined that the mammalian ortholog of MUS312 is BTBD12. Orthology between these proteins and S. cerevisiae Slx4 helped identify a conserved interaction with a second structure-specific endonuclease, SLX1. Genetic and biochemical evidence described here and in related papers suggest that MUS312 and BTBD12 direct Holliday junction resolution by at least two distinct endonucleases in different recombination and repair contexts.

DOG-1 Is the Caenorhabditis elegans BRIP1/FANCJ Homologue and Functions in Interstrand Cross-Link Repair

Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by defective DNA interstrand cross-link (ICL) repair. Here, we show that DOG-1 is the Caenorhabditis elegans homologue of FANCJ, a helicase mutated in FA-J patients. DOG-1 performs a conserved role in ICL repair, as dog-1 mutants are hypersensitive to ICL-inducing agents, but not to UVC irradiation or X rays. Genetic analysis indicated that dog-1 is epistatic with fcd-2 (C. elegans FANCD2) but is nonepistatic with brc-1 (C. elegans BRCA1), thus establishing the existence of two distinct pathways of ICL repair in worms. Furthermore, DOG-1 is dispensable for FCD-2 and RAD-51 focus formation, suggesting that DOG-1 operates downstream of FCD-2 and RAD-51 in ICL repair. DOG-1 was previously implicated in poly(G)/poly(C) (G/C) tract maintenance during DNA replication. G/C tracts remain stable in the absence of ATL-1, CLK-2 (FA pathway activators), FCD-2, BRC-2, and MLH-1 (associated FA components), implying that DOG-1 is the sole FA component required for G/C tract maintenance in a wild-type background. However, FCD-2 is required to promote deletion-free repair at G/C tracts in dog-1 mutants, consistent with a role for FA factors at the replication fork. The functional conservation between DOG-1 and FANCJ suggests a possible role for FANCJ in G/C tract maintenance in human cells.

Interstrand crosslink repair: can XPF-ERCC1 be let off the hook?

The interstrand crosslink (ICL) presents a challenge to both the cell and the scientist. From a clinical standpoint, these lesions are particularly intriguing: ICL-inducing agents are powerful tools in cancer chemotherapy, and spontaneous ICLs have recently been linked with accelerated aging phenotypes. Nevertheless, the ICL repair process has proven difficult to elucidate. Here we discuss recent additions to the current model and argue that the endonuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1 (XPF-ERCC1) has been heretofore misplaced. During nucleotide excision repair, XPF-ERCC1 makes a single-strand nick adjacent to the lesion. XPF-ERCC1 has been thought to play an analogous role in ICL repair. However, recent data has implicated XPF-ERCC1 in homologous recombination. We suggest that this role, rather than its function in nucleotide excision repair, defines its importance to ICL repair.