RESUMO
Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.
Assuntos
Microscopia , Neurônios , Animais , Interneurônios , Camundongos , Microscopia/métodos , Neurônios/fisiologia , Fótons , VigíliaRESUMO
Recent advances in single-molecule imaging of mRNAs in fixed and living cells have enabled the lives of mRNAs to be studied with unprecedented spatial and temporal detail. These approaches have moved beyond simply being able to observe specific events and have begun to allow an understanding of how regulation is coupled between steps in the mRNA life cycle. Additionally, these methodologies are now being applied in multicellular systems and animals to provide more nuanced insights into the physiological regulation of RNA metabolism.
Assuntos
RNA Mensageiro , Animais , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , DNA/ultraestrutura , Regulação da Expressão Gênica , RNA Mensageiro/ultraestrutura , Pequeno RNA não Traduzido/ultraestrutura , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , Replicação do DNA , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Humanos , Microscopia de Fluorescência , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Coloração e Rotulagem/métodos , Telômero/metabolismo , Telômero/ultraestrutura , Transcrição GênicaRESUMO
Ribosome assembly is an efficient but complex and heterogeneous process during which ribosomal proteins assemble on the nascent rRNA during transcription. Understanding how the interplay between nascent RNA folding and protein binding determines the fate of transcripts remains a major challenge. Here, using single-molecule fluorescence microscopy, we follow assembly of the entire 3' domain of the bacterial small ribosomal subunit in real time. We find that co-transcriptional rRNA folding is complicated by the formation of long-range RNA interactions and that r-proteins self-chaperone the rRNA folding process prior to stable incorporation into a ribonucleoprotein (RNP) complex. Assembly is initiated by transient rather than stable protein binding, and the protein-RNA binding dynamics gradually decrease during assembly. This work questions the paradigm of strictly sequential and cooperative ribosome assembly and suggests that transient binding of RNA binding proteins to cellular RNAs could provide a general mechanism to shape nascent RNA folding during RNP assembly.
Assuntos
Dobramento de RNA , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Modelos Biológicos , Conformação de Ácido Nucleico , Ligação Proteica , Estabilidade de RNA , RNA Ribossômico/química , Transcrição GênicaRESUMO
CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.
Assuntos
Actinomycetales/enzimologia , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/química , DNA Viral/metabolismo , Multimerização Proteica , Imagem Individual de MoléculaRESUMO
The ability to visualize and quantitatively measure dynamic biological processes in vivo and at high spatiotemporal resolution is of fundamental importance to experimental investigations in developmental biology. Light-sheet microscopy is particularly well suited to providing such data, since it offers exceptionally high imaging speed and good spatial resolution while minimizing light-induced damage to the specimen. We review core principles and recent advances in light-sheet microscopy, with a focus on concepts and implementations relevant for applications in developmental biology. We discuss how light-sheet microcopy has helped advance our understanding of developmental processes from single-molecule to whole-organism studies, assess the potential for synergies with other state-of-the-art technologies, and introduce methods for computational image and data analysis. Finally, we explore the future trajectory of light-sheet microscopy, discuss key efforts to disseminate new light-sheet technology, and identify exciting opportunities for further advances.
Assuntos
Biologia do Desenvolvimento/métodos , Microscopia de Fluorescência/tendências , Animais , Simulação por Computador , Compressão de Dados , Desenvolvimento Embrionário , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos , Análise Espaço-TemporalRESUMO
CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and â¼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA. PAPERCLIP.
Assuntos
Proteínas de Ligação a DNA/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ligação Proteica , Análise de Sequência de DNA/métodos , Sistemas CRISPR-Cas , Ensaio de Desvio de Mobilidade Eletroforética , Microscopia de Fluorescência , Motivos de NucleotídeosRESUMO
The structural characterization of protein complexes in their native environment is challenging but crucial for understanding the mechanisms that mediate cellular processes. We developed an integrative approach to reconstruct the 3D architecture of protein complexes in vivo. We applied this approach to the exocyst, a hetero-octameric complex of unknown structure that is thought to tether secretory vesicles during exocytosis with a poorly understood mechanism. We engineered yeast cells to anchor the exocyst on defined landmarks and determined the position of its subunit termini at nanometer precision using fluorescence microscopy. We then integrated these positions with the structural properties of the subunits to reconstruct the exocyst together with a vesicle bound to it. The exocyst has an open hand conformation made of rod-shaped subunits that are interlaced in the core. The exocyst architecture explains how the complex can tether secretory vesicles, placing them in direct contact with the plasma membrane.
Assuntos
Exocitose , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Complexo de Golgi/metabolismo , Modelos Moleculares , Vesículas Secretórias/metabolismoRESUMO
Cellular senescence is a stress-response mechanism implicated in various physiological processes, diseases, and aging. Current detection approaches have partially addressed the issue of senescent cell identification in clinical specimens. Effective methodologies enabling precise isolation or live tracking of senescent cells are still lacking. In-depth analysis of truly senescent cells is, therefore, an extremely challenging task. We report (1) the synthesis and validation of a fluorophore-conjugated, Sudan Black-B analog (GLF16), suitable for in vivo and in vitro analysis of senescence by fluorescence microscopy and flow cytometry and (2) the development and application of a GLF16-carrying micelle vector facilitating GLF16 uptake by living senescent cells in vivo and in vitro. The compound and the applied methodology render isolation of senescent cells an easy, rapid, and precise process. Straightforward nanocarrier-mediated GLF16 delivery in live senescent cells comprises a unique tool for characterization of senescence at an unprecedented depth.
Assuntos
Senescência Celular , Indicadores e Reagentes , Citometria de FluxoRESUMO
Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular Mtb replication induces the death of the infected macrophages and the release of bacterial aggregates. Here, we show that these aggregates can evade phagocytosis by killing macrophages in a contact-dependent but uptake-independent manner. We use time-lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytosolic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb ESX-1 secretion system, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface-exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that blocking ESX-1-mediated secretion of the EsxA/EsxB virulence factors does not eliminate the uptake-independent killing of macrophages and that the 50-kDa isoform of the ESX-1-secreted protein EspB can mediate killing in the absence of EsxA/EsxB secretion. Treatment with an ESX-1 inhibitor reduces uptake-independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti-phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung.
Assuntos
Mycobacterium tuberculosis , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Macrófagos/metabolismo , Fatores de Virulência/metabolismoRESUMO
Biological phase transitions form membrane-less organelles that generate distinct cellular environments. How molecules are partitioned between these compartments and the surrounding cellular space and the functional consequence of this localization is not well understood. Here, we report the localization of mRNA to stress granules (SGs) and processing bodies (PBs) and its effect on translation and degradation during the integrated stress response. Using single mRNA imaging in living human cells, we find that the interactions of mRNAs with SGs and PBs have different dynamics, very few mRNAs directly move between SGs and PBs, and that specific RNA-binding proteins can anchor mRNAs within these compartments. During recovery from stress, we show that mRNAs that were within SGs and PBs are translated and degraded at similar rates as their cytosolic counterparts. Our work provides a framework for using single-molecule measurements to directly investigate the molecular mechanisms of phase-separated compartments within their cellular environment.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Hibridização in Situ Fluorescente , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Estresse Fisiológico , Autoantígenos/genética , Autoantígenos/metabolismo , Transporte Biológico , Grânulos Citoplasmáticos/genética , Células HeLa , Humanos , Microscopia de Fluorescência , Ligação Proteica , Sequência de Oligopirimidina na Região 5' Terminal do RNA , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fatores de Tempo , Antígeno SS-BRESUMO
Ribosomal frameshifting during the translation of RNA is implicated in human disease and viral infection. While previous work has uncovered many details about single RNA frameshifting kinetics in vitro, little is known about how single RNA frameshift in living systems. To confront this problem, we have developed technology to quantify live-cell single RNA translation dynamics in frameshifted open reading frames. Applying this technology to RNA encoding the HIV-1 frameshift sequence reveals a small subset (â¼8%) of the translating pool robustly frameshift. Frameshifting RNA are translated at similar rates as non-frameshifting RNA (â¼3 aa/s) and can continuously frameshift for more than four rounds of translation. Fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide insight into retroviral frameshifting and could lead to alternative strategies to perturb the process in living cells.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , HIV-1/genética , Fases de Leitura Aberta , Osteoblastos/metabolismo , RNA Viral/genética , Imagem Individual de Molécula/métodos , Pareamento de Bases , Linhagem Celular Tumoral , HIV-1/metabolismo , Humanos , Modelos Genéticos , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos/síntese química , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Osteoblastos/virologia , RNA Viral/química , RNA Viral/metabolismo , Coloração e Rotulagem/métodosRESUMO
Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromossomos de Insetos/genética , Drosophila melanogaster/genética , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microscopia de Fluorescência/métodos , RNA/genética , Análise de Célula Única/métodos , Transcrição Gênica , Ativação Transcricional , Animais , Ciclo Celular/genética , Cromatina/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ Fluorescente , RNA/biossínteseRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) has emerged as a central target for cancer therapies due to the ability of PARP inhibitors to specifically kill tumors deficient for DNA repair by homologous recombination. Upon DNA damage, PARP1 quickly binds to DNA breaks and triggers ADP-ribosylation signaling. ADP-ribosylation is important for the recruitment of various factors to sites of damage, as well as for the timely dissociation of PARP1 from DNA breaks. Indeed, PARP1 becomes trapped at DNA breaks in the presence of PARP inhibitors, a mechanism underlying the cytotoxitiy of these inhibitors. Therefore, any cellular process influencing trapping is thought to impact PARP inhibitor efficiency, potentially leading to acquired resistance in patients treated with these drugs. There are numerous ADP-ribosylation targets after DNA damage, including PARP1 itself as well as histones. While recent findings reported that the automodification of PARP1 promotes its release from the DNA lesions, the potential impact of other ADP-ribosylated proteins on this process remains unknown. Here, we demonstrate that histone ADP-ribosylation is also crucial for the timely dissipation of PARP1 from the lesions, thus contributing to cellular resistance to PARP inhibitors. Considering the crosstalk between ADP-ribosylation and other histone marks, our findings open interesting perspectives for the development of more efficient PARP inhibitor-driven cancer therapies.
Assuntos
ADP-Ribosilação , Histonas , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Histonas/metabolismo , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
Most mammalian cells have molecular circadian clocks that generate widespread rhythms in transcript and protein abundance. While circadian clocks are robust to fluctuations in the cellular environment, little is known about the mechanisms by which the circadian period compensates for fluctuating metabolic states. Here, we exploit the heterogeneity of single cells both in circadian period and a metabolic parameter-protein stability-to study their interdependence without the need for genetic manipulation. We generated cells expressing key circadian proteins (CRYPTOCHROME1/2 (CRY1/2) and PERIOD1/2 (PER1/2)) as endogenous fusions with fluorescent proteins and simultaneously monitored circadian rhythms and degradation in thousands of single cells. We found that the circadian period compensates for fluctuations in the turnover rates of circadian repressor proteins and uncovered possible mechanisms using a mathematical model. In addition, the stabilities of the repressor proteins are circadian phase dependent and correlate with the circadian period in a phase-dependent manner, in contrast to the prevailing model.
Assuntos
Ritmo Circadiano , Criptocromos , Proteínas Circadianas Period , Análise de Célula Única , Proteínas Circadianas Period/metabolismo , Proteínas Circadianas Period/genética , Ritmo Circadiano/fisiologia , Criptocromos/metabolismo , Criptocromos/genética , Animais , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Relógios Circadianos/fisiologia , Humanos , Camundongos , Estabilidade ProteicaRESUMO
Fluorescence microscopy is essential for studying living cells, tissues and organisms. However, the fluorescent light that switches on fluorescent molecules also harms the samples, jeopardizing the validity of results - particularly in techniques such as super-resolution microscopy, which demands extended illumination. Artificial intelligence (AI)-enabled software capable of denoising, image restoration, temporal interpolation or cross-modal style transfer has great potential to rescue live imaging data and limit photodamage. Yet we believe the focus should be on maintaining light-induced damage at levels that preserve natural cell behaviour. In this Opinion piece, we argue that a shift in role for AIs is needed - AI should be used to extract rich insights from gentle imaging rather than recover compromised data from harsh illumination. Although AI can enhance imaging, our ultimate goal should be to uncover biological truths, not just retrieve data. It is essential to prioritize minimizing photodamage over merely pushing technical limits. Our approach is aimed towards gentle acquisition and observation of undisturbed living systems, aligning with the essence of live-cell fluorescence microscopy.
Assuntos
Inteligência Artificial , Software , Microscopia de FluorescênciaRESUMO
Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development through controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of the plant dynamin-related protein 2 (DRP2) family (hereafter DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to recruiters of dynamins, such as endophilin and amphiphysin, in CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that, despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Dinaminas , Endocitose , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clatrina/metabolismo , Clatrina/genética , Dinaminas/metabolismo , Dinaminas/genética , Endocitose/genética , Proteínas de Ligação ao GTP , Mutação/genéticaRESUMO
Huntington's disease is caused by an abnormally long polyglutamine tract in the huntingtin protein. This leads to the generation and deposition of N-terminal exon1 fragments of the protein in intracellular aggregates. We combined electron tomography and quantitative fluorescence microscopy to analyze the structural and material properties of huntingtin exon1 assemblies in mammalian cells, in yeast, and in vitro. We found that huntingtin exon1 proteins can form reversible liquid-like assemblies, a process driven by huntingtin's polyQ tract and proline-rich region. In cells and in vitro, the liquid-like assemblies converted to solid-like assemblies with a fibrillar structure. Intracellular phase transitions of polyglutamine proteins could play a role in initiating irreversible pathological aggregation.
Assuntos
Proteína Huntingtina/química , Doença de Huntington/patologia , Peptídeos/química , Transição de Fase , Agregação Patológica de Proteínas/patologia , Éxons , Células HEK293 , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Saccharomyces cerevisiaeRESUMO
Cells need to reliably control their proteome composition to maintain homeostasis and regulate growth. How protein synthesis and degradation interplay to control protein expression levels remains unclear. Here, we combined a tandem fluorescent timer and pulse-chase protein labeling to disentangle how protein synthesis and degradation control protein homeostasis in single live mouse embryonic stem cells. We discovered substantial cell-cycle dependence in protein synthesis rates and stabilization of a large number of proteins around cytokinesis. Protein degradation rates were highly variable between cells, co-varied within individual cells for different proteins, and were positively correlated with synthesis rates. This suggests variability in proteasome activity as an important source of global extrinsic noise in gene expression. Our approach paves the way toward understanding the complex interplay of synthesis and degradation processes in determining protein levels of individual mammalian cells.
Assuntos
Imagem Óptica/métodos , Proteostase/fisiologia , Animais , Ciclo Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Camundongos , Biossíntese de Proteínas/fisiologia , Proteólise , Proteoma/metabolismo , Proteômica/métodos , Análise de Célula Única/métodosRESUMO
Cellular eukaryotic replication initiation helicases are first loaded as head-to-head double hexamers on double-stranded (ds) DNA origins and then initiate S-phase DNA melting during licensed (once per cell cycle) replication. Merkel cell polyomavirus (MCV) large T (LT) helicase oncoprotein similarly binds and melts its own 98-bp origin but replicates multiple times in a single cell cycle. To examine the actions of this unlicensed viral helicase, we quantitated multimerization of MCV LT molecules as they assembled on MCV DNA origins using real-time single-molecule microscopy. MCV LT formed highly stable double hexamers having 17-fold longer mean lifetime (τ, >1,500 s) on DNA than single hexamers. Unexpectedly, partial MCV LT assembly without double-hexamer formation was sufficient to melt origin dsDNA as measured by RAD51, RPA70, or S1 nuclease cobinding. DNA melting also occurred with truncated MCV LT proteins lacking the helicase domain, but was lost from a protein without the multimerization domain that could bind only as a monomer to DNA. SV40 polyomavirus LT also multimerized to the MCV origin without forming a functional hexamer but still melted origin DNA. MCV origin melting did not require ATP hydrolysis and occurred for both MCV and SV40 LT proteins using the nonhydrolyzable ATP analog, adenylyl-imidodiphosphate (AMP-PNP). LT double hexamers formed in AMP-PNP, and melted DNA, consistent with direct LT hexamer assembly around single-stranded (ss) DNA without the energy-dependent dsDNA-to-ssDNA melting and remodeling steps used by cellular helicases. These results indicate that LT multimerization rather than helicase activity is required for origin DNA melting during unlicensed virus replication.