XPA tumor variant leads to defects in NER that sensitize cells to cisplatin.

Nucleotide excision repair (NER) reduces efficacy of treatment with platinum (Pt)-based chemotherapy by removing Pt lesions from DNA. Previous study has identified that missense mutation or loss of the NER genes Excision Repair Cross Complementation Group 1 and 2 (ERCC1 and ERCC2) leads to improved patient outcomes after treatment with Pt-based chemotherapies. Although most NER gene alterations found in patient tumors are missense mutations, the impact of mutations in the remaining nearly 20 NER genes is unknown. Towards this goal, we previously developed a machine learning strategy to predict genetic variants in an essential NER protein, Xeroderma Pigmentosum Complementation Group A (XPA), that disrupt repair. In this study, we report in-depth analyses of a subset of the predicted variants, including in vitro analyses of purified recombinant protein and cell-based assays to test Pt agent sensitivity in cells and determine mechanisms of NER dysfunction. The most NER deficient variant Y148D had reduced protein stability, weaker DNA binding, disrupted recruitment to damage, and degradation. Our findings demonstrate that tumor mutations in XPA impact cell survival after cisplatin treatment and provide valuable mechanistic insights to improve variant effect prediction. Broadly, these findings suggest XPA tumor variants should be considered when predicting chemotherapy response.

Trabectedin derails transcription-coupled nucleotide excision repair to induce DNA breaks in highly transcribed genes.

Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, anagent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, an understanding of the mechanism of the drug's TC-NER-dependent toxicity is needed. Here, we determine that abortive TC-NER of trabectedin-DNA adducts forms persistent single-strand breaks (SSBs) as the adducts block the second of the two sequential NER incisions. We map the 3'-hydroxyl groups of SSBs originating from the first NER incision at trabectedin lesions, recording TC-NER on a genome-wide scale. Trabectedin-induced SSBs primarily occur in transcribed strands of active genes and peak near transcription start sites. Frequent SSBs are also found outside gene bodies, connecting TC-NER to divergent transcription from promoters. This work advances the use of trabectedin for precision oncology and for studying TC-NER and transcription.

Detection of oxaliplatin- and cisplatin-DNA lesions requires different global genome repair mechanisms that affect their clinical efficacy.

The therapeutic efficacy of cisplatin and oxaliplatin depends on the balance between the DNA damage induction and the DNA damage response of tumor cells. Based on clinical evidence, oxaliplatin is administered to cisplatin-unresponsive cancers, but the underlying molecular causes for this tumor specificity are not clear. Hence, stratification of patients based on DNA repair profiling is not sufficiently utilized for treatment selection. Using a combination of genetic, transcriptomics and imaging approaches, we identified factors that promote global genome nucleotide excision repair (GG-NER) of DNA-platinum adducts induced by oxaliplatin, but not by cisplatin. We show that oxaliplatin-DNA lesions are a poor substrate for GG-NER initiating factor XPC and that DDB2 and HMGA2 are required for efficient binding of XPC to oxaliplatin lesions and subsequent GG-NER initiation. Loss of DDB2 and HMGA2 therefore leads to hypersensitivity to oxaliplatin but not to cisplatin. As a result, low DDB2 levels in different colon cancer cells are associated with GG-NER deficiency and oxaliplatin hypersensitivity. Finally, we show that colon cancer patients with low DDB2 levels have a better prognosis after oxaliplatin treatment than patients with high DDB2 expression. We therefore propose that DDB2 is a promising predictive marker of oxaliplatin treatment efficiency in colon cancer.

Alkylation of nucleobases by 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) sensitizes PARP1-deficient tumors.

Targeting BRCA1- and BRCA2-deficient tumors through synthetic lethality using poly(ADP-ribose) polymerase inhibitors (PARPi) has emerged as a successful strategy for cancer therapy. PARPi monotherapy has shown excellent efficacy and safety profiles in clinical practice but is limited by the need for tumor genome mutations in BRCA or other homologous recombination genes as well as the rapid emergence of resistance. In this study, we identified 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) as a small molecule that selectively kills PARP1- and xeroderma pigmentosum A-deficient cells. CDEAH is a monofunctional alkylating agent that preferentially alkylates guanine nucleobases, forming DNA adducts that can be removed from DNA by either a PARP1-dependent base excision repair or nucleotide excision repair. Treatment of PARP1-deficient cells leads to the formation of strand breaks, an accumulation of cells in S phase and activation of the DNA damage response. Furthermore, CDEAH selectively inhibits PARP1-deficient xenograft tumor growth compared to isogenic PARP1-proficient tumors. Collectively, we report the discovery of an alkylating agent inducing DNA damage that requires PARP1 activity for repair and acts synergistically with PARPi.

XPA tumor variants lead to defects in NER that sensitize cells to cisplatin.

Nucleotide excision repair (NER) neutralizes treatment with platinum (Pt)-based chemotherapy by removing Pt lesions from DNA. Previous study has identified that missense mutation or loss of either of the NER genes Excision Repair Cross Complementation Group 1 and 2 ( ERCC1 and ERCC2 ) leads to improved patient outcomes after treatment with Pt-based chemotherapies. Although most NER gene alterations found in patient tumors are missense mutations, the impact of such mutations in the remaining nearly 20 NER genes is unknown. Towards this goal, we previously developed a machine learning strategy to predict genetic variants in an essential NER scaffold protein, Xeroderma Pigmentosum Complementation Group A (XPA), that disrupt repair activity on a UV-damaged substrate. In this study, we report in-depth analyses of a subset of the predicted NER-deficient XPA variants, including in vitro analyses of purified recombinant protein and cell-based assays to test Pt agent sensitivity in cells and determine mechanisms of NER dysfunction. The most NER deficient variant Y148D had reduced protein stability, weaker DNA binding, disrupted recruitment to damage, and degradation resulting from tumor missense mutation. Our findings demonstrate that tumor mutations in XPA impact cell survival after cisplatin treatment and provide valuable mechanistic insights to further improve variant effect prediction efforts. More broadly, these findings suggest XPA tumor variants should be considered when predicting patient response to Pt-based chemotherapy. Significance: A destabilized, readily degraded tumor variant identified in the NER scaffold protein XPA sensitizes cells to cisplatin, suggesting that XPA variants can be used to predict response to chemotherapy.

MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1.

DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.

Development of Comprehensive Ultraperformance Liquid Chromatography-High-Resolution Mass Spectrometry Assays to Quantitate Cisplatin-Induced DNA-DNA Cross-Links.

Cisplatin (CP) is a common antitumor drug that is used to treat many solid tumors. The activity of CP is attributed to the formation of DNA-DNA cross-links, which consist of 1,2-intra-, 1,3-intra-, and interstrand cross-links. To better understand how each intrastrand cross-link contributes to the activity of CP, we have developed comprehensive ultraperformance liquid chromatography-selective ion monitoring (UPLC-SIM) assays to quantify 1,2-GG-, 1,2-AG-, 1,3-GCG-, and 1,3-GTG-intrastrand cross-links. The limit of quantitation for the developed assays ranged from 5 to 50 fmol or as low as 6 cross-links per 108 nucleotides. To demonstrate the utility of the UPLC-SIM assays, we first performed in vitro cross-link formation kinetics experiments. We confirmed that the 1,2-GG-intrastrand cross-links were the most abundant intrastrand cross-link and formed at a faster rate compared to 1,2-AG- and 1,3-intrastrand cross-links. Furthermore, we investigated the repair kinetics of intrastrand cross-links in CP-treated wild-type and nucleotide excision repair (NER)-deficient U2OS cells. We observed a slow decrease of both 1,2- and 1,3-intrastrand cross-links in wild-type cells and no evidence of direct repair in the NER-deficient cells. Taken together, we have demonstrated that our assays are capable of accurately quantifying intrastrand cross-links in CP-treated samples and can be utilized to better understand the activity of CP.

A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair.

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

EEPD1 promotes repair of oxidatively-stressed replication forks.

Unrepaired oxidatively-stressed replication forks can lead to chromosomal instability and neoplastic transformation or cell death. To meet these challenges cells have evolved a robust mechanism to repair oxidative genomic DNA damage through the base excision repair (BER) pathway, but less is known about repair of oxidative damage at replication forks. We found that depletion or genetic deletion of EEPD1 decreases clonogenic cell survival after oxidative DNA damage. We demonstrate that EEPD1 is recruited to replication forks stressed by oxidative damage induced by H2O2 and that EEPD1 promotes replication fork repair and restart and decreases chromosomal abnormalities after such damage. EEPD1 binds to abasic DNA structures and promotes resolution of genomic abasic sites after oxidative stress. We further observed that restoration of expression of EEPD1 via expression vector transfection restores cell survival and suppresses chromosomal abnormalities induced by oxidative stress in EEPD1-depleted cells. Consistent with this, we found that EEPD1 preserves replication fork integrity by preventing oxidatively-stressed unrepaired fork fusion, thereby decreasing chromosome instability and mitotic abnormalities. Our results indicate a novel role for EEPD1 in replication fork preservation and maintenance of chromosomal stability during oxidative stress.

ZNF212 promotes genomic integrity through direct interaction with TRAIP.

TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions of TRAIP in these processes in mammalian cells are not fully understood. Here we identify the zinc finger protein 212, ZNF212, as a novel binding partner for TRAIP and find that ZNF212 colocalizes with sites of DNA damage. The recruitment of TRAIP or ZNF212 to sites of DNA damage is mutually interdependent. We show that depletion of ZNF212 causes defects in the DDR and HR-mediated repair in a manner epistatic to TRAIP. In addition, an epistatic analysis of Zfp212, the mouse homolog of human ZNF212, in mouse embryonic stem cells (mESCs), shows that it appears to act upstream of both the Neil3 and Fanconi anemia (FA) pathways of ICLs repair. We find that human ZNF212 interacted directly with NEIL3 and promotes its recruitment to ICL lesions. Collectively, our findings identify ZNF212 as a new factor involved in the DDR, HR-mediated repair and ICL repair though direct interaction with TRAIP.

Two interaction surfaces between XPA and RPA organize the preincision complex in nucleotide excision repair.

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

XPC-PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair.

Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.

Transcriptional Perturbations of 2,6-Diaminopurine and 2-Aminopurine.

2,6-Diaminopurine (Z) is a naturally occurring adenine (A) analog that bacteriophages employ in place of A in their genetic alphabet. Recent discoveries of biogenesis pathways of Z in bacteriophages have stimulated substantial research interest in this DNA modification. Here, we systematically examined the effects of Z on the efficiency and fidelity of DNA transcription. Our results showed that Z exhibited no mutagenic yet substantial inhibitory effects on transcription mediated by purified T7 RNA polymerase and by human RNA polymerase II in HeLa nuclear extracts and in human cells. A structurally related adenine analog, 2-aminopurine (2AP), strongly blocked T7 RNA polymerase but did not impede human RNA polymerase II in vitro or in human cells, where no mutant transcript could be detected. The lack of mutagenic consequence and the presence of a strong blockage effect of Z on transcription suggest a role of Z in transcriptional regulation. Z is also subjected to removal by transcription-coupled nucleotide-excision repair (TC-NER), but not global-genome NER in human cells. Our findings provide new insight into the effects of Z on transcription and its potential biological functions.

Active DNA damage eviction by HLTF stimulates nucleotide excision repair.

Nucleotide excision repair (NER) counteracts the onset of cancer and aging by removing helix-distorting DNA lesions via a "cut-and-patch"-type reaction. The regulatory mechanisms that drive NER through its successive damage recognition, verification, incision, and gap restoration reaction steps remain elusive. Here, we show that the RAD5-related translocase HLTF facilitates repair through active eviction of incised damaged DNA together with associated repair proteins. Our data show a dual-incision-dependent recruitment of HLTF to the NER incision complex, which is mediated by HLTF's HIRAN domain that binds 3'-OH single-stranded DNA ends. HLTF's translocase motor subsequently promotes the dissociation of the stably damage-bound incision complex together with the incised oligonucleotide, allowing for an efficient PCNA loading and initiation of repair synthesis. Our findings uncover HLTF as an important NER factor that actively evicts DNA damage, thereby providing additional quality control by coordinating the transition between the excision and DNA synthesis steps to safeguard genome integrity.

A combination of direct reversion and nucleotide excision repair counters the mutagenic effects of DNA carboxymethylation.

Distinct cellular DNA damage repair pathways maintain the structural integrity of DNA and protect it from the mutagenic effects of genotoxic exposures and processes. The occurrence of O6-carboxymethylguanine (O6-CMG) has been linked to meat consumption and hypothesized to contribute to the development of colorectal cancer. However, the cellular fate of O6-CMG is poorly characterized and there is contradictory data in the literature as to how repair pathways may protect cells from O6-CMG mutagenicity. To better address how cells detect and remove O6-CMG, we evaluated the role of two DNA repair pathways in counteracting the accumulation and toxic effects of O6-CMG. We found that cells deficient in either the direct repair protein O6-methylguanine-DNA methyltransferase (MGMT), or key components of the nucleotide excision repair (NER) pathway, accumulate higher levels O6-CMG DNA adducts than wild type cells. Furthermore, repair-deficient cells were more sensitive to carboxymethylating agents and displayed an increased mutation rate. These findings suggest that a combination of direct repair and NER circumvent the effects O6-CMG DNA damage.

Polycarcin V induces DNA-damage response and enables the profiling of DNA-binding proteins.

To maintain genomic integrity and avoid diseases, the DNA-damage response (DDR) not only detects and repairs DNA lesions, but also contributes to the resistance to DNA-damaging chemotherapeutics. Targeting the DDR plays a significant role in drug discovery using the principle of synthetic lethality. The incomplete current knowledge of the DDR encouraged us to develop new strategies to identify and study its components and pathways. Polycarcin V, belonging to the C-aryl glycoside natural products, is a light-activatable DNA-intercalating agent that causes DNA damage by forming a covalent [2+2] cycloadduct with thymine residue under 365-450 nm of light irradiation in a DNA-sequence-independent manner. Taking advantage of the light-activatable feature and temporal control of DDR, we designed and synthesized polycarcin V-based bifunctional chemical probes, including one that cross-links DNA to DNA-binding protein to explore the DDR induced by polycarcin V and uncover novel DNA-protein interactions. Utilizing this chemical probe and activity-based protein profiling-stable isotope labeling with amino acids in cell culture, we identified 311 DNA-binding protein candidates, including known DDR factors and additional proteins that may be of interest in discovering new biology. We validated our approach by showing that our probe could specifically cross-link proteins involved in nucleotide excision repair (NER) that repair bulky DNA adducts. Our studies showed that the [2+2] cycloadduct formed by polycarcin V could indeed be repaired by NER in vivo. As a DNA-damaging agent, polycarcin V or its drug-like derivative plus blue light showed promising properties for psoriasis treatment, suggesting that it may itself hold promise for clinic applications.

Nucleotide excision repair leaves a mark on chromatin: DNA damage detection in nucleosomes.

Global genome nucleotide excision repair (GG-NER) eliminates a broad spectrum of DNA lesions from genomic DNA. Genomic DNA is tightly wrapped around histones creating a barrier for DNA repair proteins to access DNA lesions buried in nucleosomal DNA. The DNA-damage sensors XPC and DDB2 recognize DNA lesions in nucleosomal DNA and initiate repair. The emerging view is that a tight interplay between XPC and DDB2 is regulated by post-translational modifications on the damage sensors themselves as well as on chromatin containing DNA lesions. The choreography between XPC and DDB2, their interconnection with post-translational modifications such as ubiquitylation, SUMOylation, methylation, poly(ADP-ribos)ylation, acetylation, and the functional links with chromatin remodelling activities regulate not only the initial recognition of DNA lesions in nucleosomes, but also the downstream recruitment and necessary displacement of GG-NER factors as repair progresses. In this review, we highlight how nucleotide excision repair leaves a mark on chromatin to enable DNA damage detection in nucleosomes.

PARP Inhibition in Prostate Cancer With Homologous Recombination Repair Alterations.

PURPOSE: With the broad use of next-generation sequencing assays, it has become clear that mutations in DNA repair genes are more commonly found than previously reported. In advanced prostate cancer patients with BRCA1/2 or ATM mutations, poly (ADP-ribose) polymerase inhibition (PARPi) causes an increased overall survival advantage compared with patients without these mutations. This review explores the advantages and limitations of PARPi treatment and its use beyond BRCA1/2-altered tumors. Furthermore, it discusses the benefits of current biomarkers and what role functional biomarkers and organoids may play in addressing the involvement of homologous recombination repair mutations in tumor development and progression. METHODS: A systematic review was conducted in MEDLINE, National Library of Medicine, and ClinicalTrials.gov to identify studies published between January 1, 2016, and August 31, 2021. The search strategy incorporated terms for PARPi, BRCA, DNA damage, homologous recombination, organoids, patient-derived organoids, biomarker AND prostate cancer, breast cancer, ovarian cancer. RESULTS: A total of 261 records remained after duplicate removal, 69 of which were included in the qualitative synthesis. CONCLUSION: To improve the outcome of targeted therapy and increase sensitivity of tumor detection, patients should be repeatedly screened for DNA repair gene alterations and biomarkers. Future clinical studies should explore the use of PARPi beyond BRCA1/2 mutations and focus on finding new synthetically lethal interactions.

New Synthetic Analogs of Nitrogen Mustard DNA Interstrand Cross-Links and Their Use to Study Lesion Bypass by DNA Polymerases.

Nitrogen mustards are a widely used class of antitumor agents that exert their cytotoxic effects through the formation of DNA interstrand cross-links (ICLs). Despite being among the first antitumor agents used, the biological responses to NM ICLs remain only partially understood. We have previously reported the generation of NM ICL mimics by incorporation of ICL precursors into DNA using solid-phase synthesis at defined positions, followed by a double reductive amination reaction. However, the structure of these mimics deviated from the native NM ICLs. Using further development of our approach, we report a new class of NM ICL mimics that only differ from their native counterpart by substitution of dG with 7-deaza-dG at the ICL. Importantly, this approach allows for the synthesis of diverse NM ICLs, illustrated here with a mimic of the adduct formed by chlorambucil. We used the newly generated ICLs in reactions with replicative and translesion synthesis DNA polymerase to demonstrate their stability and utility for functional studies. These new NM ICLs will allow for the further characterization of the biological responses to this important class of antitumor agents.

The complexity and regulation of repair of alkylation damage to nucleic acids.

DNA damaging agents have been a cornerstone of cancer therapy for nearly a century. The discovery of many of these chemicals, particularly the alkylating agents, are deeply entwined with the development of poisonous materials originally intended for use in warfare. Over the last decades, their anti-proliferative effects have focused on the specific mechanisms by which they damage DNA, and the factors involved in the repair of such damage. Due to the variety of aberrant adducts created even for the simplest alkylating agents, numerous pathways of repair are engaged as a defense against this damage. More recent work has underscored the role of RNA damage in the cellular response to these agents, although the understanding of their role in relation to established DNA repair pathways is still in its infancy. In this review, we discuss the chemistry of alkylating agents, the numerous ways in which they damage nucleic acids, as well as the specific DNA and RNA repair pathways which are engaged to counter their effects.