AP endonuclease 1: Biological updates and advances in activity analysis.

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1, APEX1, REF1, HAP1) is an abasic site-specific endonuclease holding critical roles in numerous biological functions including base excision repair, the DNA damage response, redox regulation of transcription factors, RNA processing, and gene regulation. Pathologically, APE1 expression and function is linked with numerous human diseases including cancer, highlighting the importance of sensitive and quantitative assays to measure APE1 activity. Here, we summarize biochemical and biological roles for APE1 and expand on the discovery of APE1 inhibitors.

C-terminal residues of DNA polymerase β and E3 ligase required for ubiquitin-linked proteolysis of oxidative DNA-protein crosslinks.

Free radicals produce in DNA a large variety of base and deoxyribose lesions that are corrected by the base excision DNA repair (BER) system. However, the C1'-oxidized abasic residue 2-deoxyribonolactone (dL) traps DNA repair lyases in covalent DNA-protein crosslinks (DPC), including the core BER enzyme DNA polymerase beta (Polβ). Polβ-DPC are rapidly processed in mammalian cells by proteasome-dependent digestion.

Poly-ADP-ribosylation dynamics, signaling, and analysis.

ADP-ribosylation is a reversible post-translational modification that plays a role as a signaling mechanism in various cellular processes. This modification is characterized by its structural diversity, highly dynamic nature, and short half-life. Hence, it is tightly regulated at many levels by cellular factors that fine-tune its formation, downstream signaling, and degradation that together impacts cellular outcomes.

Oncometabolite 2-hydroxyglutarate suppresses basal protein levels of DNA polymerase beta that enhances alkylating agent and PARG inhibition induced cytotoxicity.

Mutations in isocitrate dehydrogenase isoform 1 (IDH1) are primarily found in secondary glioblastoma (GBM) and low-grade glioma but are rare in primary GBM. The standard treatment for GBM includes radiation combined with temozolomide, an alkylating agent. Fortunately, IDH1 mutant gliomas are sensitive to this treatment, resulting in a more favorable prognosis.

Deoxyuridine-rich cytoplasmic DNA antagonizes STING-dependent innate immune responses and sensitizes resistant tumors to anti-PD-L1 therapy.

Unlabelled: DNA damage and cytoplasmic DNA induce type-1 interferon (IFN-1) and potentiate responses to immune checkpoint inhibitors. Our prior work found that inhibitors of the DNA damage response kinase ATR (ATRi) induce IFN-1 and deoxyuridine (dU) incorporation by DNA polymerases, akin to antimetabolites. Whether and how dU incorporation is required for ATRi-induced IFN-1 signaling is not known.

CHK1 inhibitor induced PARylation by targeting PARG causes excessive replication and metabolic stress and overcomes chemoresistance in ovarian cancer.

Chemoresistance contributes to the majority of deaths in women with ovarian cancer (OC). Altered DNA repair and metabolic signaling is implicated in mediating therapeutic resistance. DNA damage checkpoint kinase 1 (CHK1) integrates cell cycle and DNA repair in replicating cells, and its inhibition causes replication stress, repair deficiency and cell cycle dysregulation.

Protein lipoylation: mitochondria, cuproptosis, and beyond.

Protein lipoylation, a crucial post-translational modification (PTM), plays a pivotal role in mitochondrial function and emerges as a key player in cell death through cuproptosis. This novel copper-driven cell death pathway is activated by excessive copper ions binding to lipoylated mitochondrial proteins, disrupting energy production and causing lethal protein aggregation and cell death. The intricate relationship among protein lipoylation, cellular energy metabolism, and cuproptosis offers a promising avenue for regulating essential cellular functions.

TRIP12 governs DNA Polymerase β involvement in DNA damage response and repair.

The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, present a challenge for genome integrity maintenance as this requires the engagement of different DNA repair pathways. Specific 'repair controllers' that facilitate DNA repair pathway crosstalk between double strand break (DSB) repair and base excision repair (BER), and regulate BER protein trafficking at lesion sites, have yet to be identified. We find that DNA polymerase β (Polβ), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partners with UBR5 and restrains DSB repair signaling.

Mouse models to explore the biological and organismic role of DNA polymerase beta.

Gene knock-out (KO) mouse models for DNA polymerase beta (Polβ) revealed that loss of Polβ leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET-mediated demethylation, more long-lived mouse models continue to be developed to further define its organismic role. The Polb-KO mouse was the first of the Cre-mediated tissue-specific KO mouse models.

Overexpression of the WWE domain of RNF146 modulates poly-(ADP)-ribose dynamics at sites of DNA damage.

Protein poly-ADP-ribosylation (PARylation) is a post-translational modification formed by transfer of successive units of ADP-ribose to target proteins to form poly-ADP-ribose (PAR) chains. PAR plays a critical role in the DNA damage response (DDR) by acting as a signaling platform to promote the recruitment of DNA repair factors to the sites of DNA damage that bind via their PAR-binding domains (PBDs). Several classes of PBD families have been recognized, which identify distinct parts of the PAR chain.

Impact of polβ/XRCC1 Interaction Variants on the Efficiency of Nick Sealing by DNA Ligase IIIα in the Base Excision Repair Pathway.

Base excision repair (BER) requires a coordination from gap filling by DNA polymerase (pol) β to subsequent nick sealing by DNA ligase (LIG) IIIα at downstream steps of the repair pathway. X-ray cross-complementing protein 1 (XRCC1), a non-enzymatic scaffolding protein, forms repair complexes with polβ and LIGIIIα. Yet, the impact of the polβ mutations that affect XRCC1 interaction and protein stability on the repair pathway coordination during nick sealing by LIGIIIα remains unknown.

A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1.

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPy ("SAR by X-ray Poses Quickly") platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe.

Co-creation and engagement in a DNA integrity cohort study.

Introduction: The partnership between a research community engagement team (CE Team) and a community advisory board (CAB) formed the basis for bidirectional communication in developing resources for participant recruitment in a DNA integrity study. Engaging with a minoritized community, this partnership focused on respect, accessibility, and expanded engagement.

Methods: A ten-member CAB, working in two groups defined by meeting time convenience, provided insight and feedback to the CE Team in the creation of recruitment and consent materials, via an iterative design process in which one CAB group reviewed and enhanced materials, and the second group tested and refined them further.

A multi-scale map of protein assemblies in the DNA damage response.

The DNA damage response (DDR) ensures error-free DNA replication and transcription and is disrupted in numerous diseases. An ongoing challenge is to determine the proteins orchestrating DDR and their organization into complexes, including constitutive interactions and those responding to genomic insult. Here, we use multi-conditional network analysis to systematically map DDR assemblies at multiple scales.

Targeting HSF1 for cancer treatment: mechanisms and inhibitor development.

Heat Shock Factor 1 (HSF1) is a master regulator of heat shock responsive signaling. In addition to playing critical roles in cellular heat shock response, emerging evidence suggests that HSF1 also regulates a non-heat shock responsive transcriptional network to handle metabolic, chemical, and genetic stress. The function of HSF1 in cellular transformation and cancer development has been extensively studied in recent years.

Polβ/XRCC1 heterodimerization dictates DNA damage recognition and basal Polβ protein levels without interfering with mouse viability or fertility.

DNA Polymerase β (Polβ) performs two critical enzymatic steps during base excision repair (BER) - gap filling (nucleotidyl transferase activity) and gap tailoring (dRP lyase activity). X-ray repair cross complementing 1 (XRCC1) facilitates the recruitment of Polβ to sites of DNA damage through an evolutionarily conserved Polβ/XRCC1 interaction interface, the V303 loop. While previous work describes the importance of the Polβ/XRCC1 interaction for human Polβ protein stability and recruitment to sites of DNA damage, the impact of disrupting the Polβ/XRCC1 interface on animal viability, physiology, and fertility is unknown.

Quantitative Analysis of Nuclear Poly(ADP-Ribose) Dynamics in Response to Laser-Induced DNA Damage.

Poly(ADP-ribose) (PAR), catalyzed by members of the poly(ADP-ribose) polymerase family of enzymes, is a posttranslational modification with a critical role in most mechanisms of DNA repair. Upon activation of poly(ADP-ribose) polymerase isoforms 1 and 2 (PARP-1 and PARP-2), the proteins of the base excision repair (BER) and single-strand break repair (SSBR) pathways form DNA lesion-dependent, transient complexes to facilitate repair. PAR is central to the temporal dynamics of BER/SSBR complex assembly and disassembly.

A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage.

The Comet or single-cell gel electrophoresis assay is a highly sensitive method to measure cellular, nuclear genome damage. However, low throughput can limit its application for large-scale studies. To overcome these limitations, a 96-well CometChip platform was recently developed that increases throughput and reduces variation due to simultaneous processing and automated analysis of 96 samples.

Live Cell Detection of Poly(ADP-Ribose) for Use in Genetic and Genotoxic Compound Screens.

Poly(ADP-ribose) (PAR) is a molecular scaffold that aids in the formation of DNA repair protein complexes. Tools to sensitively quantify PAR in live cells have been lacking. We recently described the LivePAR probe (EGFP fused to the RNF146-encoded WWE PAR binding domain) to measure PAR formation at sites of laser micro-irradiation in live cells.

Overcoming Temozolomide Resistance in Glioblastoma via Enhanced NAD Bioavailability and Inhibition of Poly-ADP-Ribose Glycohydrolase.

Glioblastoma multiforme (GBM) is an incurable brain cancer with an average survival of approximately 15 months. Temozolomide (TMZ) is a DNA alkylating agent for the treatment of GBM. However, at least 50% of the patients treated with TMZ show poor response, primarily due to elevated expression of the repair protein O-methylguanine-DNA methyltransferase (MGMT) or due to defects in the mismatch repair (MMR) pathway.