DNA Interstrand Research Articles

Polyamines alleviate the inhibitory effect of the DNA cross-linking agent mitomycin C on root growth

ABSTRACTPolyamines (putrescine, spermidine and spermine) are ubiquitously present in various types of cells of living organisms. They are involved in a variety of cellular processes, including cell proliferation and cell differentiation, and are required for abiotic stress tolerances in plants. However, it is still not understood whether polyamines are involved in the plant growth inhibition caused by DNA-damaging agents. In this study, we examined the effects of polyamines on the inhibition of plant root growth and gene expression in Arabidopsis thaliana treated with mitomycin C (MMC), a genotoxic agent that induces DNA interstrand crosslinks. We found that polyamines alleviated the inhibitory effect caused by MMC on root growth. In addition, we also found that polyamines alleviated the increased expression of AtBRCA1 and AtRAD51 genes induced by MMC treatment. Our study provides the first evidence that polyamines contribute to tolerance against plant-growth inhibition caused by a DNA-damaging chemical.

Abstract 591: Improving Cardiovascular Health Through DNA Repair

Accumulated DNA damage and oxidative stress play a critical role in the etiology of cardiovascular diseases. Malfunctions of human DNA repair proteins can lead to increased myocardial infarctions, ischemic heart disease, and congestive heart failure. The FANCJ helicase and the BRCA1 tumor suppressor are essential proteins that coordinate the repair of interstrand DNA crosslinks and double-stranded breaks in human cells. Their activities are especially needed in heart muscle, which undergoes tremendous physiological and chemical stress. Our limited understanding of how FANCJ and BRCA1 target damaged DNA structures remains a major barrier to research progress. As an independent investigator, my primary focus is to assess the impact of oxidative DNA damage on cardiovascular health through systematic dissection of the DNA repair pathways maintained by FANCJ and BRCA1. A detailed understanding of these molecular mechanisms would provide new diagnostic and therapeutic strategies to detect and to treat ischemic heart disease and congestive heart failure. Here, we have examined the DNA binding properties of FANCJ and BRCA1 using fluorescence spectroscopy and biolayer interferometry. We have also measured the viabilities of human cardiomyocytes and probed their sensitivity to reactive oxygen species by automated cell counting and single-cell electrophoresis. The DNA binding results, which describe precisely how FANCJ and BRCA1 organize onto damaged DNA sites, are compared with the cell-based analysis to correlate their DNA interaction patterns with cardioprotection. Our future work will utilize this platform to predict the risks of FANCJ and BRCA1 single-nucleotide polymorphisms that still have unknown cardiovascular effects.

FANCD2 Binding to H4K20me2 via a Methyl-Binding Domain Is Essential for Efficient DNA Cross-Link Repair.

Fanconi anemia (FA) is an inherited disease characterized by bone marrow failure and increased cancer risk. FA is caused by mutation of any 1 of 22 genes, and the FA proteins function cooperatively to repair DNA interstrand cross-links (ICLs). A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins, which occurs within chromatin. How FANCD2 and FANCI are anchored to chromatin remains unknown. In this study, we identify and characterize a FANCD2 histone-binding domain (HBD) and embedded methyl-lysine-binding domain (MBD) and demonstrate binding specificity for H4K20me2. Disruption of the HBD/MBD compromises FANCD2 chromatin binding and nuclear focus formation and its ability to promote error-free DNA interstrand cross-link repair, leading to increased error-prone repair and genome instability. Our study functionally describes the first FA protein chromatin reader domain and establishes an important link between this human genetic disease and chromatin plasticity.

Targeting DNA Repair in Tumor Cells via Inhibition of ERCC1-XPF.

The ERCC1-XPF heterodimer is a 5'-3' structure-specific endonuclease, which plays an essential role in several DNA repair pathways in mammalian cells. ERCC1-XPF is primarily involved in the repair of chemically induced helix-distorting and bulky DNA lesions, such as cyclobutane pyrimidine dimers (CPDs), and DNA interstrand cross-links. Inhibition of ERCC1-XPF has been shown to potentiate cytotoxicity of platinum-based drugs and cyclophosphamide in cancer cells. In this study, the previously described ERCC1-XPF inhibitor 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-methylpiperazin-1-yl)methyl)phenol (compound 1) was used as a reference compound. Following the outcome of docking-based virtual screening (VS), we synthesized seven novel derivatives of 1 that were identified in silico as being likely to have high binding affinity for the ERCC1-XPF heterodimerization interface by interacting with the XPF double helix-hairpin-helix (HhH2) domain. Two of the new compounds, 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-cyclohexylpiperazin-1-yl)methyl)phenol (compound 3) and 4-((6-chloro-2-methoxyacridin-9-yl)amino)-2-((4-(2-(dimethylamino)ethyl) piperazin-1-yl) methyl) phenol (compound 4), were shown to be potent inhibitors of ERCC1-XPF activity in vitro. Compound 4 showed significant inhibition of the removal of CPDs in UV-irradiated cells and the capacity to sensitize colorectal cancer cells to UV radiation and cyclophosphamide.

DNA cross-link repair safeguards genomic stability during premeiotic germ cell development.

Germline de novo mutations are the basis of evolutionary diversity but also of genetic disease. However, the molecular origin, mechanisms and timing of germline mutagenesis are not fully understood. Here, we define a fundamental role for DNA interstrand crosslink repair in the germline. This repair process is essential for primordial germ cell (PGC) maturation during embryonic development. Inactivation of crosslink repair leads to genetic instability that is restricted to PGCs within the genital ridge during a narrow temporal window. Having successfully activated the PGC transcriptional program, a potent quality control mechanism detects and drives damaged PGCs into apoptosis. These findings therefore define a source of DNA damage and the nature of the subsequent DNA repair response in germ cells, which ensures faithful transmission of the genome between generations.

Abstract 4750: The role of drug transporters in the acquired resistance to PBD dimer-containing antibody drug conjugates

Abstract Antibody drug conjugates (ADCs) delivering interstrand cross-linking pyrrolobenzodiazepine (PBD) dimers are being evaluated clinically in both hematological and solid tumors. These include ADCT-301, ADCT-402, MEDI3726 and rovalpituzumab tesirine (Rova-T) that contain the payload tesirine which releases the PBD dimer warhead SG3199. An important consideration in the future development of PBD ADCs is the development of clinical acquired resistance. The aim was to generate and characterize in vitro acquired resistant cell lines in both a hematological and solid tumor setting. To generate acquired resistant cells to ADCT-301 (targeting CD25) or warhead SG3199, CD25-positive Karpas-299 cells were incubated with an approximate GI50 dose of the drug for 96 h. Cells were washed and returned to fresh medium until normal cell growth was restored. This was repeated until a stable acquired resistance was established. The level of resistance achieved was >700-fold for the ADC, and 3-fold for the naked PBD. Cross-resistance between the agents was observed and also with an ADC delivering a different PBD dimer. Similarly, resistant cells to ADCT-502 (targeting HER2) or SG3199 were established using HER2-positive NCI-N87 cells incubated with GI50 doses for 144 h. The level of resistance achieved was 8 and 4-fold, respectively, and again, cross-resistance was observed. DNA interstrand cross-linking was measured in the parental and resistant lines treated with equi-molar doses of ADC or SG3199. In every case, the resistant lines produced fewer cross-links indicating an up-stream mechanism of resistance preventing SG3199 from reaching its DNA target. In the Karpas ADCT-301-resistant line, a small (15%) decrease in CD25 cell surface expression could not account for the level of resistance. RT2 Profiler PCR Array for human drug-transporters was used to probe the cell lines. In the Karpas ADCT-301-resistant line three gene products were significantly upregulated: ABCG2 (168-fold), SLCO2A1 (4.6) and ABCC2 (3.5). Significantly upregulated in the Karpas SG3199-resistant cells were SLCO2B1 (15.4), ABCC12 (6.4), ATP7A (4.6), SLC16A2 (2.8), ABCG2 (2.8), SLC7A9 (2.5), ABCB4 (2.3) and ABCC11 (2.1). Upregulation of ABCG2, ABCC2 and SLCO2B1 was confirmed by RT-PCR. In NCI-N87 ADCT-502-resistant cells, ABCG2 (91), SLC22A3 (65), ABCC2 (43), SLCO2B1 (30) and SLC7A7 (6.8) and in NCI-N87 SG3199-resistant cells ABCG2 (115), SLC22A3 (61), SLCO2B1 (54), ABCC2 (36), SLC7A7 (8), AQP7 (6.4) and SLC238A3 (2.9) were upregulated. Upregulation of ABCG2, ABCC2, SLCO2B1, SLC7A7 and SLC22A3 was confirmed by RT-PCR and for ABCG2 and ABCC2 also by western blot. ABCB1(MDR1) was not upregulated in any of the lines. These data show that acquired resistance to both PBD-ADCs and SG3199 can involve specific drug transporters. This has clinical implications as potential biomarkers of resistance and for the rational design of drug combinations. Citation Format: Simon Corbett, Francesca Zammarchi, Philip W Howard, Patrick H van Berkel, John A Hartley. The role of drug transporters in the acquired resistance to PBD dimer-containing antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4750.

Abstract 3852: Development of1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine hybrids as potent anticancer agents to small cell lung cancer

Abstract Angiogenesis is a hallmark of cancer development, especially in lung cancer and colorectal cancer. Unfortunately, the rapid development of resistance to anti-angiogenesis agents has limited the clinical benefits for the treatment. To improve the efficacy of anti-angiogenesis agents, we designed and synthesized a series of new hybrid molecules that are composed of benzophthalazine (anti-angiogenesis moiety) and bis(hydroxymethyl)pyrrole (DNA interstrand crosslinking moiety). We demonstrate that these compounds are the dual function inducing DNA crosslinking and anti-angiogenesis. 1,2-Bis(hydroxymethyl)benzopyrrolo[2,1-a]phthalazine derivatives displayed potent cytotoxicity to leukemia and a batch of solid tumor cell lines. Among them, small cell lung cancer (SCLC) cells were the most susceptible cells with the IC50 values ranged from dozens of nanomolar to submicromolar. Significantly, we observed total tumor remission by treatment of SCLC H526 (TP53 WT) xenografts with formulated compound 8 at the dose of 20mg/kg, 5 times/week for 2 weeks. In addition, compound 8 was also more potent than cisplatin in suppression of growth of SCLC H211 (TP53 mutant) xenografts. Combination treatment of H211 cells with compound 8 and cisplatin displayed the synergistically cell killing. Our preliminary results further showed that co-treatment of compound 8 and low dose cisplatin significantly suppressed tumor growth and prolonged the survival rate of mice bearing H211 xenografts. In contract to cisplatin, treatment of mice with compound 8 did not cause body weight lost, indicating low systematic toxicity of compound 8. In summary, our results indicate that bis(hydroxymethyl)benzopyrrolo[2,1-a]phthalazine hybrids, either alone or in combination with other therapeutic drugs, are potent anticancer agents against SCLC and deserve for future development. Note: This abstract was not presented at the meeting. Citation Format: Tai-Lin Chen, Vicky Jain, Yi-Wen Lin, Tsann-Long Su, Te-Chang Lee. Development of1,2-bis(hydroxymethyl)benzo[g]pyrrolo[2,1-a]phthalazine hybrids as potent anticancer agents to small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3852.

Targeting the High-Mobility Group Box 3 Protein Sensitizes Chemoresistant Ovarian Cancer Cells to Cisplatin.

Chemotherapeutic regimens for ovarian cancer often include the use of DNA interstrand crosslink-inducing agents (e.g., platinum drugs) or DNA double-strand break-inducing agents. Unfortunately, the majority of patients fail to maintain a durable response to treatment, in part, due to drug resistance, contributing to a poor survival rate. In this study, we report that cisplatin sensitivity can be restored in cisplatin-resistant ovarian cancer cells by targeting the chromatin-associated high-mobility group box 3 (HMGB3) protein. HMGB proteins have been implicated in the pathogenesis and prognosis of ovarian cancer, and HMGB3 is often upregulated in cancer cells, making it a potential selective target for therapeutic intervention. Depletion of HMGB3 in cisplatin-sensitive and cisplatin-resistant cells resulted in transcriptional downregulation of the kinases ATR and CHK1, which attenuated the ATR/CHK1/p-CHK1 DNA damage signaling pathway. HMGB3 was associated with the promoter regions of ATR and CHK1, suggesting a new role for HMGB3 in transcriptional regulation. Furthermore, HMGB3 depletion significantly increased apoptosis in cisplatin-resistant A2780/CP70 cells after cisplatin treatment. Taken together, our results indicate that targeted depletion of HMGB3 attenuates cisplatin resistance in human ovarian cancer cells, increasing tumor cell sensitivity to platinum drugs. SIGNIFICANCE: This study shows that targeting HMGB3 is a potential therapeutic strategy to overcome chemoresistance in ovarian cancer.

A Fanci knockout mouse model reveals common and distinct functions for FANCI and FANCD2.

Fanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci −/− mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci−/− Fancd2−/− also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.

CTDP1 regulates breast cancer survival and DNA repair through BRCT-specific interactions with FANCI

BRCA1 C-terminal domains are found in a specialized group of 23 proteins that function in the DNA damage response to protect genomic integrity. C-terminal domain phosphatase 1 (CTDP1) is the only phosphatase with a BRCA1 C-terminal domain in the human proteome, yet direct participation in the DNA damage response has not been reported. Examination of the CTDP1 BRCA1 C-terminal domain-specific protein interaction network revealed 103 high confidence interactions enriched in DNA damage response proteins, including FANCA and FANCI that are central to the Fanconi anemia DNA repair pathway necessary for the resolution of DNA interstrand crosslink damage. CTDP1 expression promotes DNA damage-induced FANCA and FANCD2 foci formation and enhances homologous recombination repair efficiency. CTDP1 was found to regulate multiple aspects of FANCI activity, including chromatin localization, interaction with γ-H2AX, and SQ motif phosphorylations. Knockdown of CTDP1 increases MCF-10A sensitivity to DNA interstrand crosslinks and double-strand breaks, but not ultraviolet radiation. In addition, CTDP1 knockdown impairs in vitro and in vivo growth of breast cancer cell lines. These results elucidate the molecular functions of CTDP1 in Fanconi anemia interstrand crosslink repair and identify this protein as a potential target for breast cancer therapy.

Interstrand DNA Cross-Links Derived from Reaction of a 2-Aminopurine Residue with an Abasic Site.

Efficient methods for the site-specific installation of structurally defined interstrand cross-links in duplex DNA may be useful in a wide variety of fields. The work described here developed a high-yield synthesis of chemically stable interstrand cross-links resulting from a reductive amination reaction between an abasic site and the noncanonical nucleobase 2-aminopurine in duplex DNA. Results from footprinting, liquid chromatography-mass spectrometry, and stability studies support the formation of an N2-alkylamine attachment between the 2-aminopurine residue and the Ap site. The reaction performs best when the 2-aminopurine residue on the opposing strand is offset 1 nt to the 5'-side of the abasic site. The cross-link confers substantial resistance to thermal denaturation (melting). The cross-linking reaction is fast (complete in 4 h), employs only commercially available reagents, and can be used to generate cross-linked duplexes in sufficient quantities for biophysical, structural, and DNA repair studies.

HSF2BP Interacts with a Conserved Domain of BRCA2 and Is Required for Mouse Spermatogenesis

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability, and DNA interstrand crosslink repair in vertebrates. We identify HSF2BP, a protein previously described as testis specific and not characterized functionally, as an interactor of BRCA2 in mouse embryonic stem cells, where the 2 proteins form a constitutive complex. HSF2BP is transcribed in all cultured human cancer cell lines tested and elevated in some tumor samples. Inactivation of the mouse Hsf2bp gene results in male infertility due to a severe HR defect during spermatogenesis. The BRCA2-HSF2BP interaction is highly evolutionarily conserved and maps to armadillo repeats in HSF2BP and a 68-amino acid region between the BRC repeats and the DNA binding domain of human BRCA2 (Gly2270-Thr2337) encoded by exons 12 and 13. This region of BRCA2 does not harbor known cancer-associated missense mutations and may be involved in the reproductiverather than the tumor-suppressing function of BRCA2.

EVALUATING THE EFFECTIVENESS OF THE TUMOR-INITIATING STEM CELLS ERADICATION STRATEGY ON THE EXAMPLE OF HUMAN GLIOBLASTOMA CELL LINE U87

The effectiveness of the new therapeutic approach aimed at the destruction of the cancer cell community was evaluated on the immortalized human glioblastoma cell line U87. Initially, the reference elements of a new strategy for eradication of tumor-initiating stem cells were characterized. The main points of the strategy were as follows. 1) Evaluation of the TAMRA+ (tumor-initiating stem cells) presence in the population of U87 culture cells. 2) Determination of the reference time points of the DNA interstrand cross-links repair, induced by cross-linking cytostatics mitomycin C. 3) Determination of the day after initiation of therapy when TAMRA+ cells accumulate and are synchronously present in the G1/S sensitive for the treatment phase of the cell cycle. Based on the data obtained, a therapeutic regimen aimed at eradicating TAMRA+ cells (tumor-initiating stem cells) was identified. Treatment of the culture was carried out by cross-linking cytostatic mitomycin C and complex DNA preparation. Transplantation experiments showed that high experimental doses of mitomycin C (20 μg/ml) totally destroy transplantation potential of U87 cells. Diminution of mitomycin C to therapeutic doses (5 μg/ ml) clearly demonstrated effect of complex double-stranded DNA preparation to reducing of U87 tumorigenic potential. These fully confirmed the concept of developed cancer treatment technology.

WIP1 Contributes to the Adaptation of Fanconi Anemia Cells to DNA Damage as Determined by the Regulatory Network of the Fanconi Anemia and Checkpoint Recovery Pathways.

DNA damage adaptation (DDA) allows the division of cells with unrepaired DNA damage. DNA repair deficient cells might take advantage of DDA to survive. The Fanconi anemia (FA) pathway repairs DNA interstrand crosslinks (ICLs), and deficiencies in this pathway cause a fraction of breast and ovarian cancers as well as FA, a chromosome instability syndrome characterized by bone marrow failure and cancer predisposition. FA cells are hypersensitive to ICLs; however, DDA might promote their survival. We present the FA-CHKREC Boolean Network Model, which explores how FA cells might use DDA. The model integrates the FA pathway with the G2 checkpoint and the checkpoint recovery (CHKREC) processes. The G2 checkpoint mediates cell-cycle arrest (CCA) and the CHKREC activates cell-cycle progression (CCP) after resolution of DNA damage. Analysis of the FA-CHKREC network indicates that CHKREC drives DDA in FA cells, ignoring the presence of unrepaired DNA damage and allowing their division. Experimental inhibition of WIP1, a CHKREC component, in FA lymphoblast and cancer cell lines prevented division of FA cells, in agreement with the prediction of the model.

The Biochemical Role of the Human NEIL1 and NEIL3 DNA Glycosylases on Model DNA Replication Forks.

Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.

Roles of High Mobility Group Box Proteins in Nucleotide Excision Repair‐associated Processing of DNA Interstrand Crosslinks

Many traditional anti‐cancer chemotherapeutic agents induce DNA interstrand crosslinks (ICLs), lesions that form covalent bonds between the DNA strands. ICLs are highly cytotoxic if not properly repaired because they can block basic cellular processes such as transcription and replication. Nucleotide excision repair (NER) is a critical repair mechanism in processing ICLs in human cells. In addition to NER, ICLs are also processed through DNA double‐strand break repair pathways (e.g. homologous recombination) in S‐phase. We have previously demonstrated that the high mobility group box protein (HMGB1), a non‐histone architectural protein, can bind to site‐directed ICLs with high affinity and modulate error‐free repair processing of such lesions. In addition, we have found that HMGB1 associates with the NER damage recognition protein XPA in human cells and facilitates the recruitment of XPA to the damaged site. Further, we have demonstrated that HMGB1 can architecturally modify the damaged substrate by inducing negative supercoils, which may facilitate DNA damage processing by NER. We hypothesize that HMGB1 can modulate NER processing of ICLs by aiding NER damage‐recognition factors in locating damaged DNA. To test this hypothesis we are using a mutation‐reporter system to measure mutation frequencies and spectra generated by site‐directed ICLs in human cells. There are two NER subpathways that recognize DNA damage via different mechanisms. I am investigating in which pathway(s) HMGB1 is involved by utilizing various cell lines that are proficient or deficient for an essential recognition protein in each pathway. Additionally, we are investigating if HMGB2 and HMGB3, protein family members that are highly similar in structure to HMGB1, are also co‐factors in NER. Preliminary evidence suggests that HMGB1 may be involved in transcription coupled‐NER of site‐directed ICLs. If future evidence supports these data, this would indicate that HMGB1 could associate with transcription machinery and aid in recruiting other NER co‐factors, such as XPA. Future genetic, molecular biological, and biochemical assays will provide additional insight as to the roles of the HMGB proteins in ICL repair. The processing and repair of ICLs in human cells is not completely understood, and therefore a detailed study to define the molecular mechanisms of ICL processing is warranted. Identifying new proteins involved in ICL processing may lead to new pharmacological targets, which may help us to improve the outcome of cancer treatment.Support or Funding InformationNIH/NCI to K.V. (CA093729) and NSF GRFP to J.G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Photo-induced DNA interstrand cross-links formed by a coumarin-modified nucleoside

Coumarins are a class of naturally occurring compounds that have been shown to form photochemical DNA interstrand cross-links (ICLs). However, study of a coumarin base has not been explored. Using nucleophilic substitution and phosphoramidite chemistry, we synthesized a coumarin base-containing oligonucleotide. Upon exposure to long-wave ultraviolet light, the coumarin-modified oligonucleotide formed ICLs with complementary oligonucleotide containing dT and dC opposite the coumarin base, presumably through a [2 + 2] cycloaddition mechanism. Moderate yields with both bases were observed; though, dT has a higher reactivity than dC. Overall, this work provides new means for biochemical characterization of ICLs formed by coumarins.

TRAIP is a master regulator of DNA interstrand crosslink repair.

Summary:Cells often utilize multiple pathways to repair the same DNA lesion, and pathway choice has profound implications for the fidelity of genome maintenance. DNA interstrand cross-links (ICLs) block DNA replication and transcription by covalently linking the two strands of DNA, and the cytotoxicity of ICLs is exploited for chemotherapy. In Xenopus egg extracts, replication fork collision with ICLs initiates two distinct repair pathways. The NEIL3 glycosylase can cleave the cross-link1, but if this fails, the Fanconi anemia (FA) proteins incise the phosphodiester backbone surrounding the ICL, generating a double-strand break (DSB) intermediate that is repaired by homologous recombination2. How the simpler NEIL3 pathway is prioritized over the FA pathway, which can cause genomic rearrangements, is unknown. Here, we show that the E3 ubiquitin ligase TRAIP regulates both pathways. TRAIP appears to associate with the leading edge of the replisome, ubiquitylating any protein in the replisome’s path, including the replicative DNA helicase CMG (CDC45-MCM2-7-GINS) when two replisomes converge at an ICL. In this setting, short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for CMG unloading by the p97 ATPase, enabling the FA pathway. Our results identify TRAIP as a master regulator of replisome stability and ICL repair pathway choice.

High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2.

Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-strand breaks (DSB), which subsequently hindered the recruitment of the downstream protein Rad51 to DSB in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for effective ICL repair. Delayed FancD2 de-ubiquitination was associated with the increased chromatin retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the response to cisplatin therapies.

Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway.

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.