Biology subjects

Van Houten, B.

Publications and source records attributed to Van Houten, B..

11 recordsLinked to original sources

bioRxiv2026

ATR kinase inhibitors induce mitochondrial fission in CD8+ T cells and impair immune memory in vivo

The DNA damage response kinase ATR restrains CDK1 activity during S and G2 phases of the cell cycle, confining CDK1-driven processes to mitosis. ATR kinase inhibitors were originally developed to potentiate chemotherapy-induced DNA damage at stalled replication forks and to disrupt DNA damage-induced cell cycle checkpoints. Recent evidence, however, reveals that these inhibitors also disrupt cell cycle organization in cells that have not sustained any DNA damage. We show that ATR kinase inhibitors potently trigger unscheduled mitochondrial fission, causing loss of mitochondrial mass in actively dividing CD8+ T cells that persists in memory CD8+ T cells. Moreover, ATR inhibition during the peak of CD8+ T cell expansion in a mouse model of LCMV Armstrong infection impairs the formation of immune memory. These findings carry significant clinical implications. ATR kinase inhibitors are currently being evaluated in clinical trials in combination with chemotherapy, radiation, and immune checkpoint inhibitors in patients where anti-tumor immune responses are recognized as a determinant of durable response. Our results identify an unexpected consequence of ATR inhibition that disrupts cellular metabolism with broad implications for both preclinical research and clinical application.

cancer biology↗

bioRxiv2026

Molecular basis of nick ligation in the nucleosome by DNA Ligase IIIα

Eukaryotic genomic DNA is packaged into chromatin through a fundamental repeating unit known as the nucleosome core particle. Within this chromatin context, genomic DNA is constantly exposed to endogenous and exogenous stress that result in the formation of DNA damage, which must be effectively repaired to maintain genome stability. Single-strand breaks (SSBs) are among the most prevalent forms of DNA damage that arise via the oxidation-induced disintegration of the sugar-phosphate backbone or as repair intermediates during base excision repair. DNA ligase III (LigIII) is one of the primary enzymes responsible for repairing SSBs containing an intact 5'-phosphate and 3'-OH (nick) during the terminal step of single-strand break repair (SSBR) and base excision repair (BER) pathways. To date, a complete mechanistic description for how LigIII processes nicks within chromatin remains elusive. Here, we use a combination of biochemical assays, molecular dynamics simulations, and cryogenic electron microscopy (cryo-EM) to define the molecular basis of nick ligation in the nucleosome by LigIII. Quantitative enzyme kinetics reveal that the LigIII ligation rate is highly dependent on the translational position of the nick in the nucleosome, where nicks near the nucleosome entry/exit site are ligated with moderate efficiency and nicks near the nucleosome dyad are refractory to ligation. Cryo-EM structures of LigIII bound to nicks at four unique translational positions in the nucleosome reveal the structural basis for this position-dependent catalytic activity, identifying that local steric constraints imposed by the histone octamer prevent LigIII from readily adopting a ligation-competent conformation. Further biochemical and structural analysis demonstrates that the scaffolding protein XRCC1, which forms a heterodimer with LigIII, does not substantially alter the ability of LigIII to bind or ligate nicks in the nucleosome. Together, this work provides foundational insight into the processing of nicks in the nucleosome during the terminal step of SSBR/BER.

biochemistry↗

bioRxiv2026

PARP1 recruits SPRTN to DNA-protein crosslinks through a conserved poly-ADP-ribose binding domain.

DNA-protein crosslinks (DPCs) are toxic DNA lesions formed by the covalent attachment of proteins to DNA. Failure to resolve DPCs leads to genomic instability, premature aging, and cancer predisposition. Although multiple proteases and the 26S proteasome degrade DPCs, how these lesions are detected and marked for proteolysis remains unclear. Here, we show that poly-(ADP-ribose) polymerases (PARP1/2) sense DPCs and modify them with poly(ADP-ribose) (PAR) to promote repair via a SPRTN-Tdp1 axis. We discovered a Nudix homology domain (NHD) in SPRTN that mediates direct non-covalent PAR binding and is important for DPC repair. Loss of PARP1/2 activity or mutation of the SPRTN NHD leads to sustained DPCs. Single-molecule analysis revealed that SPRTN does not bind efficiently to the DPC, however after the addition of PARP1 in the presence of NAD+, SPRTN binding to the DPC was significantly increased. Our findings establish PARP1/2 enzymes as immediate DPC sensors, reveal PARylation as a signal marking DPCs for SPRTN-dependent degradation, and identify SPRTN as the first PARP-directed protease.

molecular biology↗

bioRxiv2026

A Dynamic Search Mechanism Enables APE1 to Identify AP-Sites in DNA

Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions, arising thousands of times per cell each day. These AP-sites threaten genomic stability and, if left unrepaired, can lead to mutagenesis and human disease. The essential base excision repair enzyme Apurinic/Apyrimidinic Endonuclease I (APE1) initiates repair by cleaving DNA at AP-sites, yet how APE1 efficiently locates and recognizes these lesions within vast excesses of undamaged DNA has remained poorly understood. Using single-molecule imaging, we show that APE1 employs a dynamic search strategy that integrates 1D and 3D diffusion to rapidly scan DNA. On non-damaged DNA, APE1 undergoes fast diffusion, enabling efficient interrogation of large genomic regions within a single binding event. Upon encountering an AP-site, APE1 transitions from a mobile search state into a stationary, lesion-bound complex that is retained at the site of damage. Additional experiments with APE1 variants demonstrate that the intrinsically disordered N-terminal domain of APE1 supports 1D diffusion, whereas residue R177 stabilizes APE1 at the AP-site once recognized, and the catalytic residues D210 and E96 facilitate enzyme release after cleavage. Together, these findings define the molecular basis by which APE1 balances rapid genome surveillance with stable lesion engagement and timely release. More broadly, this work provides a mechanistic framework for how DNA repair enzymes efficiently locate and process rare lesions embedded within an excess of undamaged DNA. Significance StatementDNA repair enzymes face the formidable challenge of locating rare lesions hidden within vast stretches of undamaged DNA. The essential enzyme APE1 is tasked with identifying and initiating repair of AP-sites, a frequent DNA lesion, to maintain genome integrity. We show that APE1 overcomes this challenge by using a dynamic search mechanism that integrates rapid scanning with precise lesion recognition. Its unstructured N-terminal domain facilitates fast movement along non-damaged DNA, while specific active site residues stabilize engagement at AP-sites and release after catalysis. This coordination of diffusion, recognition, and turnover allows APE1 to efficiently survey the genome and repair AP-sites to maintain genome stability.

biochemistry↗

bioRxiv2025

Nucleosome unwrapping and PARP1 allostery drive affinities for chromatin and DNA breaks

Poly[ADP-ribose] polymerase 1 (PARP1) detects DNA strand breaks that occur in duplex DNA and chromatin. We employed correlative optical tweezers and fluorescence microscopy to quantify how single molecules of PARP1 identify single-strand breaks (i.e., nicks), undamaged nucleosome core particles (NCP) and NCPs containing DNA nicks. Fluorescently-tagged PARP1 or PARP2 from nuclear extracts bound nicks with nanomolar affinity but did not engage undamaged dsDNA regions. In contrast, PARP1 avidly bound undamaged NCPs, and partial NCP unwrapping induced by DNA tension significantly increased the on rate and affinity. Catalytically dead PARP1 or EB-47 inhibition greatly increased PARP1 affinity to DNA nicks and undamaged NCP, implicating a mechanism where PARP1 reverse allostery regulates PARP1 retention to undamaged chromatin. We also monitored ADP-ribosylation in real time upon PARP1 binding undamaged or nicked NCPs. These results provide key mechanistic insights into domain allostery and how pharmacological intervention alters PARP1 binding dynamics for therapeutic impacts.

biophysics↗

bioRxiv2024

The human Shu complex promotes RAD51 activity by modulating RPA dynamics on ssDNA.

Templated DNA repair that occurs during homologous recombination and replication stress relies on RAD51. RAD51 activity is positively regulated by BRCA2 and the RAD51 paralogs. The Shu complex is a RAD51 paralog-containing complex consisting of SWSAP1 and SWS1. We demonstrate that SWSAP1-SWS1 binds RAD51, maintains RAD51 filament stability, and enables strand exchange. Using single molecule confocal fluorescence microscopy combined with optical tweezers, we show that SWSAP1-SWS1 decorates RAD51 filaments proficient for homologous recombination. We also find SWSAP1-SWS1 enhances RPA diffusion on ssDNA. Importantly, we show human sgSWSAP1 and sgSWS1 knockout cells are sensitive to pharmacological inhibition of PARP and APE1. Lastly, we identify cancer variants in SWSAP1 that alter SWS1 complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target.

biochemistry↗

bioRxiv2023

Single-molecule analysis of purified proteins and nuclear extracts: insights from 8-oxoguanine glycosylase 1

By observing one molecule at a time, single-molecule studies can offer detailed insights about biomolecular processes including on rates, off rates, and diffusivity of molecules on strands of DNA. A recent technological advance (Single-molecule Analysis of DNA-binding proteins from Nuclear Extracts, SMADNE) has lowered the barrier to entry for single-molecule studies, and single-molecule dynamics can now be determined directly out of nuclear extracts, providing information in an intermediate environment between purified proteins in isolation and the heterogeneity of a nucleus. To compare and contrast the single-molecule DNA binding dynamics in nuclear extracts versus purified proteins, combined optical tweezers and fluorescence microscopy experiments were performed with purified GFP-tagged 8-oxoguanine glycosylase 1 (OGG1), purified GFP-OGG1 spiked into nuclear extracts, and nuclear extracts from human cells overexpressing GFP-OGG1. We observed differences in undamaged DNA binding during DNA damage search in each of the three conditions. Purified GFP-OGG1 engaged undamaged DNA for a weighted average lifetime of 5.7 s and 21% of these events underwent DNA diffusion after binding. However, unlike other glycosylases studied by SMADNE, OGG1 does not bind non-damaged DNA efficiently in nuclear extracts. In contrast, GFP-OGG1 binding dynamics on DNA substrates containing oxidative damage were relatively similar in all three conditions, with the weighted average binding lifetimes varying from 2.2 s in nuclear extracts to 7.8 s with purified GFP-OGG1 in isolation. Finally, we compared the purified protein and nuclear extract approaches for a catalytically dead OGG1 variant (GFP-OGG1-K249Q). This variant greatly increased the binding lifetime for oxidative DNA damage, with the weighted average lifetime for GFP-OGG1-249Q in nuclear extracts at 15.4 s vs 10.7 s for the purified protein. SMADNE will provide a new window of observation into the behavior of nucleic acid binding proteins only accessible by biophysicists trained in protein purification and protein labeling.

biochemistry↗

bioRxiv2023

Single-molecule analysis reveals TDG exhibits multiple modes of linear diffusion to process 5-formylcytosine.

Base excision repair is the main pathway involved in active DNA demethylation. 5-formylctyosine and 5-carboxylcytosine, two oxidized moieties of methylated cytosine, are recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. Using single molecule fluorescence experiments, we studied TDG in the presence and absence of 5-formylctyosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of a lesion. We probed TDG active site variants and truncated N-terminus revealing how these variants alter the lesion search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG was found to either bypass, colocalize with, or encounter but not bypass the nucleosome. However, truncating the N-terminus reduced the number of interactions with the nucleosome. Our findings provide unprecedented mechanistic insights into how TDG searches for DNA lesions in chromatin.

biophysics↗

bioRxiv2022

UV irradiation remodels the specificity landscape of transcription factors

Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hyper-mutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs we identified a surprising but reproducible effect at certain non-consensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and non-consensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.

biophysics↗

bioRxiv2022

Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE)

Single-molecule characterization of protein-DNA dynamics provides unprecedented mechanistic details about numerous nuclear processes. Here, we describe a new method that rapidly generates single-molecule information for fluorescently tagged proteins isolated from nuclear extracts of human cells. This approach determines binding lifetimes (koff), events per second ([~]kon), positional dependence (specificity), and characterizes 1D diffusion along DNA. We demonstrated the wide applicability of this approach on three forms of DNA damage using seven native DNA repair proteins and two structural variants, including: poly(ADP-ribose) polymerase (PARP1), heterodimeric ultraviolet-damaged DNA-binding protein (UV-DDB), xeroderma pigmentosum complementation group C protein (XPC), and 8-oxoguanine glycosylase 1 (OGG1). By measuring multiple fluorescent colors simultaneously, we additionally characterized the assembly and disassembly kinetics of multi-protein complexes on DNA. Thus, Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE) provides new insights about damage recognition and represents a universal technique that can be used to rapidly characterize numerous protein-DNA interactions.

molecular biology↗

bioRxiv2021

Telomeric 8-oxoguanine drives rapid premature senescence in the absence of telomere shortening

Oxidative stress is a primary cause of cellular senescence and contributes to the pathogenesis of numerous human diseases. Oxidative damage to telomeric DNA is proposed to trigger premature senescence by accelerating telomere shortening. Here we tested this model directly using a precision tool to produce the common base lesion 8-oxo-guanine (8oxoG) exclusively at telomeres in human fibroblast and epithelial cells. A single induction of telomeric 8oxoG is sufficient to trigger multiple hallmarks of p53-dependent senescence. Telomeric 8oxoG activates ATM and ATR signaling, and enriches for markers of telomere dysfunction in replicating, but not quiescent cells. Acute 8oxoG production fails to shorten telomeres, but rather generates fragile sites and delayed mitotic DNA synthesis at telomeres, indicative of impaired replication. Based on our results we propose that oxidative stress promotes rapid senescence by producing oxidative base lesions which drive replication-dependent telomere fragility and dysfunction in the absence of shortening and shelterin loss.

molecular biology↗