SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair

Abstract Interstrand crosslinks (ICLs) are highly toxic DNA lesions that are repaired via a complex process requiring the coordination of several DNA repair pathways. Defects in ICL repair result in Fanconi anemia, which is characterized by bone marrow failure, developmental abnormalities, and a high incidence of malignancies. SLX4, also known as FANCP, acts as a scaffold protein and coordinates multiple endonucleases that unhook ICLs, resolve homologous recombination intermediates, and perhaps remove unhooked ICLs. In this study, we explored the role of SLX4IP, a constitutive factor in the SLX4 complex, in ICL repair. We found that SLX4IP is a novel regulatory factor; its depletion sensitized cells to treatment with ICL-inducing agents and led to accumulation of cells in the G2/M phase. We further discovered that SLX4IP binds to SLX4 and XPF–ERCC1 simultaneously and that disruption of one interaction also disrupts the other. The binding of SLX4IP to both SLX4 and XPF–ERCC1 not only is vital for maintaining the stability of SLX4IP protein, but also promotes the interaction between SLX4 and XPF–ERCC1, especially after DNA damage. Collectively, these results demonstrate a new regulatory role for SLX4IP in maintaining an efficient SLX4–XPF–ERCC1 complex in ICL repair.

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On the role of FAN1 in Fanconi anemia

Blood ◽

10.1182/blood-2012-04-420604 ◽

2012 ◽

Vol 120 (1) ◽

pp. 86-89 ◽

Cited By ~ 22

Author(s):

Juan P. Trujillo ◽

Leonardo B. Mina ◽

Roser Pujol ◽

Massimo Bogliolo ◽

Joris Andrieux ◽

...

Keyword(s):

Fanconi Anemia ◽

Bone Marrow Failure ◽

Pcr Amplification ◽

Minor Role ◽

Interstrand Crosslink ◽

Genetic Features ◽

Chromosome Fragility ◽

A Minor ◽

Interstrand Crosslink Repair ◽

Crosslink Repair

Abstract Fanconi anemia (FA) is a rare bone marrow failure disorder with defective DNA interstrand crosslink repair. Still, there are FA patients without mutations in any of the 15 genes individually underlying the disease. A candidate protein for those patients, FA nuclease 1 (FAN1), whose gene is located at chromosome 15q13.3, is recruited to stalled replication forks by binding to monoubiquitinated FANCD2 and is required for interstrand crosslink repair, suggesting that mutation of FAN1 may cause FA. Here we studied clinical, cellular, and genetic features in 4 patients carrying a homozygous 15q13.3 micro-deletion, including FAN1 and 6 additional genes. Biallelic deletion of the entire FAN1 gene was confirmed by failure of 3′- and 5′-PCR amplification. Western blot analysis failed to show FAN1 protein in the patients' cell lines. Chromosome fragility was normal in all 4 FAN1-deficient patients, although their cells showed mild sensitivity to mitomycin C in terms of cell survival and G2 phase arrest, dissimilar in degree to FA cells. Clinically, there were no symptoms pointing the way to FA. Our results suggest that FAN1 has a minor role in interstrand crosslink repair compared with true FA genes and exclude FAN1 as a novel FA gene.

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Coordinated Cut and Bypass: Replication of Interstrand Crosslink-Containing DNA

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.699966 ◽

2021 ◽

Vol 9 ◽

Author(s):

Qiuzhen Li ◽

Kata Dudás ◽

Gabriella Tick ◽

Lajos Haracska

Keyword(s):

Dna Replication ◽

Fanconi Anemia ◽

Replication Fork ◽

Defense Mechanism ◽

Genome Instability ◽

Dna Lesions ◽

Fanconi Anemia Pathway ◽

Interstrand Crosslinks ◽

Dna Lesion ◽

Interstrand Crosslink

DNA interstrand crosslinks (ICLs) are covalently bound DNA lesions, which are commonly induced by chemotherapeutic drugs, such as cisplatin and mitomycin C or endogenous byproducts of metabolic processes. This type of DNA lesion can block ongoing RNA transcription and DNA replication and thus cause genome instability and cancer. Several cellular defense mechanism, such as the Fanconi anemia pathway have developed to ensure accurate repair and DNA replication when ICLs are present. Various structure-specific nucleases and translesion synthesis (TLS) polymerases have come into focus in relation to ICL bypass. Current models propose that a structure-specific nuclease incision is needed to unhook the ICL from the replication fork, followed by the activity of a low-fidelity TLS polymerase enabling replication through the unhooked ICL adduct. This review focuses on how, in parallel with the Fanconi anemia pathway, PCNA interactions and ICL-induced PCNA ubiquitylation regulate the recruitment, substrate specificity, activity, and coordinated action of certain nucleases and TLS polymerases in the execution of stalled replication fork rescue via ICL bypass.

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The yeast Hrq1 helicase stimulates Pso2 translesion nuclease activity and thereby promotes DNA interstrand crosslink repair

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013626 ◽

2020 ◽

Vol 295 (27) ◽

pp. 8945-8957 ◽

Cited By ~ 3

Author(s):

Cody M. Rogers ◽

Chun-Ying Lee ◽

Samuel Parkins ◽

Nicholas J. Buehler ◽

Sabine Wenzel ◽

...

Keyword(s):

Single Molecule ◽

Nuclease Activity ◽

Dna Lesions ◽

Principal Mechanism ◽

Single Molecule Fret ◽

Interstrand Crosslink ◽

Physical Barriers ◽

A Site ◽

Interstrand Crosslink Repair

DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)– and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro. We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.

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EXD2 and WRN exonucleases are required for interstrand crosslink repair in Drosophila

10.1101/284307 ◽

2018 ◽

Author(s):

Pratima Chennuri ◽

Lynne S. Cox ◽

Robert D. C. Saunders

Keyword(s):

Genomic Instability ◽

Fanconi Anemia ◽

Werner Syndrome ◽

Fruit Fly ◽

Chromosome Breakage ◽

Cancer Therapies ◽

Principal Mechanism ◽

Interstrand Crosslinks ◽

Interstrand Crosslink ◽

Interstrand Crosslink Repair

AbstractInterstrand crosslinks (ICLs) present a major threat to genome integrity, preventing both the correct transcription of active chromatin and complete replication of the genome. This is exploited in genotoxic chemotherapy where ICL induction is used to kill highly proliferative cancer cells. Repair of ICLs involves a complex interplay of numerous proteins, including those in the Fanconi anemia (FA) pathway, though alternative and parallel pathways have been postulated. Here, we investigate the role of the 3’-5’ exonuclease, EXD2, and the highly related WRN exonuclease (implicated in premature ageing human Werner syndrome), in repair of interstrand crosslinks in the fruit fly, Drosophila melanogaster. We find that flies mutant for EXD2 (DmEXD2) have elevated rates of genomic instability resulting from chromosome breakage and loss of the resulting acentric fragments, in contrast to WRN exonuclease (DmWRNexo) mutants where excess homologous recombination is the principal mechanism of genomic instability. Most notably, we demonstrate that proliferating larval neuroblasts mutant for either DmWRNexo or DmEXD2 are deficient in repair of DNA interstrand crosslinks caused by diepoxybutane or mitomycin C, strongly suggesting that each nuclease individually plays a role in repair of ICLs in flies. These findings have significant implications not only for understanding the complex process of ICL repair in humans, but also for enhancing cancer therapies that rely on ICL induction, with caveats for cancer therapy in Werner syndrome and Fanconi anemia patients.

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Faculty Opinions recommendation of BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.715247809.791652856 ◽

2012 ◽

Author(s):

Lee Zou

Keyword(s):

Homologous Recombination ◽

Interstrand Crosslink ◽

Interstrand Crosslink Repair ◽

Crosslink Repair

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Faculty Opinions recommendation of BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.715247809.790652813 ◽

2012 ◽

Author(s):

Junjie Chen

Keyword(s):

Homologous Recombination ◽

Interstrand Crosslink ◽

Interstrand Crosslink Repair ◽

Crosslink Repair

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Theoretical Study on the Photo-Oxidation and Photoreduction of an Azetidine Derivative as a Model of DNA Repair

Molecules ◽

10.3390/molecules26102911 ◽

2021 ◽

Vol 26 (10) ◽

pp. 2911

Author(s):

Miriam Navarrete-Miguel ◽

Antonio Francés-Monerris ◽

Miguel A. Miranda ◽

Virginie Lhiaubet-Vallet ◽

Daniel Roca-Sanjuán

Keyword(s):

Electron Transfer ◽

Energy Barrier ◽

Ring Opening ◽

Dna Lesions ◽

Barrier Heights ◽

Dna Lesion ◽

Electron Reduction ◽

The Stability ◽

Electron Transfer Processes

Photocycloreversion plays a central role in the study of the repair of DNA lesions, reverting them into the original pyrimidine nucleobases. Particularly, among the proposed mechanisms for the repair of DNA (6-4) photoproducts by photolyases, it has been suggested that it takes place through an intermediate characterized by a four-membered heterocyclic oxetane or azetidine ring, whose opening requires the reduction of the fused nucleobases. The specific role of this electron transfer step and its impact on the ring opening energetics remain to be understood. These processes are studied herein by means of quantum-chemical calculations on the two azetidine stereoisomers obtained from photocycloaddition between 6-azauracil and cyclohexene. First, we analyze the efficiency of the electron-transfer processes by computing the redox properties of the azetidine isomers as well as those of a series of aromatic photosensitizers acting as photoreductants and photo-oxidants. We find certain stereodifferentiation favoring oxidation of the cis-isomer, in agreement with previous experimental data. Second, we determine the reaction profiles of the ring-opening mechanism of the cationic, neutral, and anionic systems and assess their feasibility based on their energy barrier heights and the stability of the reactants and products. Results show that oxidation largely decreases the ring-opening energy barrier for both stereoisomers, even though the process is forecast as too slow to be competitive. Conversely, one-electron reduction dramatically facilitates the ring opening of the azetidine heterocycle. Considering the overall quantum-chemistry findings, N,N-dimethylaniline is proposed as an efficient photosensitizer to trigger the photoinduced cycloreversion of the DNA lesion model.

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