Biology subjects

Nagel, Z. D.

Publications and source records attributed to Nagel, Z. D..

10 recordsLinked to original sources

bioRxiv2026

MUTYH activity maintains telomere stability in response to chronic telomeric 8-oxoguanine damage in cancer cells

Telomeres are highly susceptible to oxidative DNA damage, particularly 8-oxoguanine (8-oxoG), which is processed by glycosylase-initiated base excision repair (BER). OGG1 removes 8-oxoG opposite C, and MUTYH removes A misinserted opposite 8-oxoG to prevent mutations. While OGG1 has an established role in telomere protection, the contribution of MUTYH to telomere stability in cancer cells after oxidative DNA damage remains poorly understood. Using a chemoptogenetic system to induce targeted 8-oxoG lesions specifically at telomeres in HeLa cancer cells, we demonstrate that MUTYH is required to prevent telomere shortening, telomere loss, and genomic instability after chronic damage. Yet, telomere damage in MUTYH-deficient cells does not cause sustained DNA damage signaling or reduced cellular proliferation. Whole-genome sequencing further reveals enrichment of G to T transversions within telomeric repeats in MUTYH-deficient cells, consistent with increased mutagenesis due to unrepaired 8-oxoG:A mispairs. Combined loss of MUTYH and OGG1 rescues damage-induced telomere aberrations and genomic instability, implicating BER-generated single-strand break (SSB) intermediates as major contributors to telomere instability. In agreement, exo-FISH and S1-END-seq analyses reveal that repair-proficient cells rapidly accumulate SSB intermediates after damage, which are later resolved, whereas glycosylase-deficient cells exhibit SSBs at later time points. Together, these findings identify MUTYH as a critical guardian of telomere integrity during chronic oxidative stress and provide insight into how defective BER at telomeres contributes to genomic instability in cancer cells, with implications for cancers associated with MUTYH deficiency and mutations.

molecular biology↗

bioRxiv2026

Ex vivo stem-like cell families model evolution of glioblastoma therapeutic resistance

Glioblastoma (GBM) arises from stem-like cells (GSCs) that exhibit intrinsic therapeutic resistance and can be positively selected by treatment, rendering recurrent GBM intractable. Mechanistic dissection of therapeutic resistance evolution has been limited by scarce matched primary/recurrent tractable models. To address this gap, we developed "resistant GSC families", a within-patient matched platform that models therapy-driven selection by ex vivo deriving, from the same primary whole GBM, temozolomide (TMZ)- and ionizing radiation (IR)-selected GSCs, alongside a treatment-naive control (CTRL-GSC). This design enables pressure-specific dissection of resistance evolution, separating chemotherapy- and radiotherapy-associated genetic alterations and adaptations that are often confounded in primary-versus-recurrent comparisons. Using this framework, we link TMZ-driven relapse-like states to either mismatch repair (MMR)-dependent stable resistance or O6-methylguanine-DNA methyltransferase (MGMT)-independent drug tolerance, and identify adaptive DNA damage response and cell-cycle changes as a route to increased radioresistance. Across pressures, treatment-emergent GSCs accumulate chromosomal alterations and exhibit adaptive phenotypic remodeling, including increased receptor tyrosine kinase activity. Resistant GSC families represent a model enabling mechanistic studies and hypothesis-driven testing of strategies aimed at preventing or treating GBM recurrence.

cancer biology↗

bioRxiv2025

MSIanalyzer: Targeted Nanopore Sequencing Enables Single Nucleotide Resolution Analysis of Microsatellite Instability Diversity

We present a targeted sequencing-based pipeline that profiles microsatellite instability (MSI) at single-nucleotide resolution. Targeted amplicons from the five widely studied Bethesda panel microsatellite loci were sequenced using Oxford Nanopore Technology in two microsatellite unstable colorectal cancer cell lines (HCT15, HCT116), two microsatellite stable cancer cell lines (TK6, U2OS), and two peripheral blood mononuclear cell samples from healthy donors. An anchor-extension algorithm was developed to capture repeat motifs while allowing interruptions, using a threshold informed by platform-specific error. Cluster-aware Dirichlet-multinomial and beta-binomial tests were applied for between-sample comparisons while accounting for read-level clustering within samples. The algorithm revealed distinct repeat profiles in HCT15 and HCT116 compared to other cell types and uncovered allelic diversity across samples at different MSI loci. Our approach complements existing short tandem repeat callers by preserving read-level diversity and delivering targeted, quantitative MSI calls with potential applications in mechanistic research and clinical assay development.

molecular biology↗

bioRxiv2025

Double strand breaks drive toxicity in Huntington's disease mice with or without somatic expansion.

There has been a substantial investment in elucidating the mechanism of expansion in hopes of identifying therapeutic targets for Huntington disease (HD). Although an expanded CAG allele is the causal mutation for HD, there is evidence that somatic expansion may not be the only disease driver. We report here that double strand breaks (DSBs) drive HD toxicity by an independent mechanism from somatic expansion. The mutant HD protein inhibits non-homologous end joining (NHEJ) activity, leading to the accumulation of DSBs. DSBs promote transcriptional pathology in mice that cannot expand their CAG tracts somatically. Conversely, Inhibition of DSBs reverses neuronal toxicity in animals that undergo somatic expansion. Although they coexist in neurons, DSBs and somatic expansion are independent therapeutic targets for HD.

molecular biology↗

bioRxiv2024

D-type cyclins regulate DNA mismatch repair in the G1 and S phases of the cell cycle, maintaining genome stability

The large majority of oxidative DNA lesions occurring in the G1 phase of the cell cycle are repaired by base excision repair (BER) rather than mismatch repair (MMR) to avoid long resections that can lead to genomic instability and cell death. However, the molecular mechanisms dictating pathway choice between MMR and BER have remained unknown. Here, we show that, during G1, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins shield p21 from its two ubiquitin ligases CRL1SKP2 and CRL4CDT2 in a CDK4/6-independent manner. In turn, p21 competes through its PCNA-interacting protein degron with MMR components for their binding to PCNA. This inhibits MMR while not affecting BER. At the G1/S transition, the CRL4AMBRA1-dependent degradation of D-type cyclins renders p21 susceptible to proteolysis. These timely degradation events allow the proper binding of MMR proteins to PCNA, enabling the repair of DNA replication errors. Persistent expression of cyclin D1 during S-phase increases the mutational burden and promotes microsatellite instability. Thus, the expression of D-type cyclins inhibits MMR in G1, whereas their degradation is necessary for proper MMR function in S. One-Sentence SummaryTo maintain genome stability, D-type cyclins limit mismatch repair (MMR) in G1, whereas their degradation is necessary for proper MMR function in S phase.

cell biology↗

bioRxiv2023

ATM phosphorylates the FATC domain of DNA-PKcs at threonine 4102 to promote non-homologous end joining

Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double strand breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Phosphorylation at T4102 stabilizes the interaction between DNA-PKcs and the Ku-DNA complex and promotes assembly and stabilization of the NHEJ machinery at DSBs. Ablating phosphorylation at this site results in decreased NHEJ, radiosensitivity, and increased radiation-induced genomic instability. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PKcs.

molecular biology↗

bioRxiv2021

An active learning framework improves tumor variant interpretation

For precision medicine to reach its full potential for treatment of cancer and other diseases, protein variant effect prediction tools are needed that characterize variants of unknown significance (VUS) in a patients genome with respect to their likelihood to influence treatment response and outcomes. However, the performance of most variant prediction tools is limited by the difficulty of acquiring sufficient training and validation data. To overcome these limitations, we applied an iterative active learning approach starting from available biochemical, evolutionary, and functional annotations. The potential of active learning to improve variant interpretation was first demonstrated by applying it to synthetic and deep mutational scanning (DMS) datasets for four cancer-relevant proteins. We then probed its utility to guide interpretation and functional validation of tumor VUS in a potential biomarker for cancer therapy sensitivity, the nucleotide excision repair (NER) protein Xeroderma Pigmentosum Complementation Group A (XPA). A quantitative high-throughput cell-based NER activity assay, fluorescence-based multiplex flow-cytometric host cell reactivation (FM-HCR), was used to validate XPA VUS selected by the active learning strategy. In all cases, selecting VUS for validation by active learning yielded an improvement in performance over traditional learning. These analyses suggest that active learning is well-suited to significantly improve interpretation of VUS and cancer patient genomes.

cancer biology↗

bioRxiv2021

Molecular basis and functional consequences of the interaction between the Base Excision Repair DNA glycosylase NEIL1 and RPA

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein replication protein A (RPA) modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd [~] 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against ionizing radiation-induced DNA lesions in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed that RPA interaction is not required for NEIL1 activity against oxidative damage in duplex DNA, and furthermore revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.

biophysics↗

bioRxiv2020

Chromatin-Bound PARP1 Correlates with Upregulation of Inflammatory Genes in Response to Long-Term Treatment with Veliparib

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat human HCC1937 BRCA1 mutant and isogenic BRCA1-complemented cells for three weeks with veliparib, a PARP inhibitor. We show that long-term treatment with veliparib results in chromatin-bound PARP1 in the BRCA1 mutant cells, and that this correlates with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/ signalling effector stimulator of interferon genes (STING) pathway. In contrast, long-term treatment of isogenic BRCA1-complemented cells with veliparib does not result in chromatin-associated PARP or significant upregulation of the inflammatory response. Our results suggest that long-term veliparib treatment may prime BRCA1 mutant tumors for positive responses to immune checkpoint blockade.

cancer biology↗

bioRxiv2019

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer

DNA repair defects have been increasingly focused on as therapeutic targets. In hormone positive breast cancer, XRCC1-deficient tumors have been identified and proposed as targets for combination therapies that damage DNA and inhibit DNA repair pathways. XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) polymerase 1, DNA polymerase {beta} (POL {beta}), and DNA ligases. Loss of XRCC1 confers BER defects and hypersensitivity to DNA damaging agents. BER defects have not been evaluated in triple negative breast cancer (TNBC), for which new therapeutic targets and therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects in TNBC, we examined XRCC1 expression and localization in the TCGA database and in TNBC cell lines. High XRCC1 expression was observed for TNBC tumors in the TCGA database and expression of XRCC1 varied between TNBC cell lines. We also observed changes in XRCC1 subcellular localization in TNBCs that alter the ability to repair base lesions and single-strand breaks. Subcellular localization changes were also observed for POL {beta} that did not correlate with XRCC1 localization. Basal levels of DNA damage were also measured in the TNBC cell lines, and damage levels correlated with observed changes in XRCC1 expression, localization, and repair functions. The results confirmed that XRCC1 expression changes may indicate DNA repair capacity changes but emphasize that basal DNA damage levels along with expression and localization are better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in TNBC preclinical models and the TCGA database, XRCC1 expression levels should be considered when evaluating treatment responses of TNBC preclinical model cells.

cancer biology↗