RESUMO
Cell death is a fundamental biological process with different modes including apoptosis and necrosis. In contrast to programmed apoptosis, necrosis was previously considered disordered and passive, but it is now being realized to be under regulation by certain biological pathways. However, the intracellular dynamics that coordinates with cellular structure changes during necrosis remains unknown, limiting our understanding of the principles of necrosis. Here, we characterized the spatiotemporal intracellular diffusion dynamics in cells undergoing necrosis, using three-dimensional single-particle tracking of quantum dots. We found temporally increased diffusion rates in necrotic cells and spatially enhanced diffusion heterogeneity in the cell periphery, which could be attributed to the reduced molecular crowding resulting from cell swelling and peripheral blebbing, respectively. Moreover, the three-dimensional intracellular diffusion transits from strong anisotropy to nearly isotropy, suggesting a remodeling of the cytoarchitecture that relieves the axial constraint on intracellular diffusion during necrosis. Our results reveal the remarkable alterations of intracellular diffusion dynamics and biophysical properties in necrosis, providing insight into the well-organized nonequilibrium necrotic cell death from a biophysical perspective.
Assuntos
Necrose , Pontos Quânticos , Pontos Quânticos/química , Humanos , Difusão , Células HeLaRESUMO
Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.
Assuntos
Trifosfato de Adenosina , DNA Helicases , RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Imagem Individual de Molécula , Terminação da Transcrição Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Polimerase II/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Imagem Individual de Molécula/métodos , RNA Helicases/metabolismo , RNA Helicases/genética , Transcrição Gênica , RNA Fúngico/metabolismo , RNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Fúngico/genética , HidróliseRESUMO
Cell migration plays important roles in many biological processes, but how migrating cells orchestrate intracellular molecules and subcellular structures to regulate their speed and direction is still not clear. Here, by characterizing the intracellular diffusion and the three-dimensional lamellipodium structures of fish keratocyte cells, we observe a strong positive correlation between the intracellular diffusion and cell migration speed and, more importantly, discover a switching of cell migration modes with reversible intracellular diffusion variation and lamellipodium structure deformation. Distinct from the normal fast mode, cells migrating in the newly-found slow mode have a deformed lamellipodium with swollen-up front and thinned-down rear, reduced intracellular diffusion and compartmentalized macromolecule distribution in the lamellipodium. Furthermore, in turning cells, both lamellipodium structure and intracellular diffusion dynamics are also changed, with left-right symmetry breaking. We propose a mechanism involving the front-localized actin polymerization and increased molecular crowding in the lamellipodium to explain how cells spatiotemporally coordinate the intracellular diffusion dynamics and the lamellipodium structure in regulating their migrations.
Assuntos
Eritrócitos Anormais , Pseudópodes , Animais , Movimento Celular , DifusãoRESUMO
Intracellular transport plays an important role in maintaining the physiological functions of cells. Here, we describe a protocol for 3D single-particle tracking within living cells. We detail the use of a two-focal imaging system and the analytical steps for quantifying 3D transport dynamics. This protocol can be used to characterize the intracellular diffusion and trafficking of macromolecules, nanoparticles, and endocytic vesicles in adherent cells. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2022).
Assuntos
Nanopartículas , Imagem Individual de Molécula , Difusão , Transporte BiológicoRESUMO
Intracellular transport, regulated by complex cytoarchitectures and active driving forces, is crucial for biomolecule translocations and relates to many cellular functions. Despite extensive knowledge obtained from two-dimensional (2D) experiments, the real three-dimensional (3D) spatiotemporal characteristics of intracellular transport is still unclear. With 3D single-particle tracking, we comprehensively studied the transport dynamics of endocytic cargos. With varying timescale, the intracellular transport changes from thermal-dominated 3D-constrained motion to active-dominated quasi-2D motion. Spatially, the lateral motion is heterogeneous with peripheral regions being faster than perinuclear regions, while the axial motion is homogeneous across the cells. We further confirmed that such anisotropy and heterogeneity of vesicle transport result from actively directed motion on microtubules. Strikingly, inside the vesicles, we observed endocytic nanoparticles make diffusive motions on their inner membranes when microtubules are absent, suggesting endocytic cargos are normally localized at the inner vesicle membranes through a physical connection to the microtubules outside during transport.
RESUMO
The histone chaperone FACT (FAcilitates Chromatin Transcription) plays an essential role in transcription and DNA replication by its dual functions on nucleosome assembly to maintain chromatin integrity and nucleosome disassembly to destabilize nucleosome and facilitate its accessibility simultaneously. Mono-ubiquitination at Lysine 119 of H2A (ubH2A) has been suggested to repress transcription by preventing the recruitment of FACT at early elongation process. However, up to date, how ubH2A directly affects FACT on nucleosome assembly and disassembly remains elusive. In this study, we demonstrated that the dual functions of FACT are differently regulated by ubH2A. The H2A ubiquitination does not affect FACT's chaperone function in nucleosome assembly and FACT can deposit ubH2A-H2B dimer on tetrasome to form intact nucleosome. However, ubH2A greatly restricts FACT binding on nucleosome and inhibits its activity of nucleosome disassembly. Interestingly, deubiquitination of ubH2A rescues the nucleosome disassembly function of FACT to activate gene transcription. Our findings provide mechanistic insights of how H2A ubiquitination affects FACT in breaking nucleosome and maintaining its integrity, which sheds light on the biological function of ubH2A and various FACT's activity under different chromatin states.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina , Camundongos , Ligação Proteica , UbiquitinaçãoRESUMO
Cell morphology and migration depend critically on the adhesions on the extracellular matrix (ECM), determined by the transmembrane protein integrins. The epithelial to mesenchymal transition (EMT) is a prominent transformation process in which adherent cells acquire a mesenchymal phenotype and a promoted migration. EMT plays important roles in embryonic development and cancer metastasis, and its hallmarks include the acquisition of front-back cell polarity and loss of cell-cell contact. However, how integrins dynamically regulate cell-ECM adhesions and cellular behaviors during EMT is still unclear. Using single-particle tracking of ß1-integrins labeled with quantum dots, the temporal-spatial on-membrane dynamics of integrins in the EMT of MCF10A cells is revealed. ß1-integrins exhibit significantly enhanced dynamics, which temporally behave more diffusive and less immobilized, and spatially become distributed asymmetrically with front regions being more dynamic. These dynamic alterations are shown to arise from microtubule remodeling in EMT. The results shed new light on the EMT mechanism from the cell-ECM adhesion perspective, and suggest that the enhanced integrin diffusion may represent as a new hallmark of EMT.
Assuntos
Transição Epitelial-Mesenquimal , Integrinas , Movimento Celular , Células Epiteliais , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Transdução de SinaisRESUMO
There is broad consensus that RecQ family helicase is a high-order oligomer that dissociates into a dimer upon ATP binding. This conclusion is based mainly on studies of highly purified recombinant proteins, and the oligomeric states of RecQ helicases in living cells remain unknown. We show here that, in contrast to current models, monomeric RECQL helicase is more abundant than oligomer/dimer forms in living cells. Further characterization of endogenous BtRECQL and isolated monomeric BtRECQL using various approaches demonstrates that both endogenous and recombinant monomeric BtRECQL effectively function as monomers, displaying higher helicase and ATPase activities than dimers and oligomers. Furthermore, monomeric BtRECQL unfolds intramolecular G-quadruplex DNA as efficiently as human RECQL and BLM helicases. These discoveries have implications for understanding endogenous RECQL oligomeric structures and their regulation. It is worth revisiting oligomeric states of the other members of the RecQ family helicases in living cells.
Assuntos
Neoplasias da Mama/metabolismo , DNA/metabolismo , Predisposição Genética para Doença/genética , RecQ Helicases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Neoplasias da Mama/genética , Bovinos , Quadruplex G , Proteínas Recombinantes/metabolismoRESUMO
Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.
Assuntos
Motivos de Aminoácidos , Candida albicans/química , Candida tropicalis/química , Domínio Catalítico , Telomerase/química , Motivos de Aminoácidos/genética , Candida albicans/enzimologia , Candida tropicalis/enzimologia , Catálise , Domínio Catalítico/genética , Cromatografia em Gel , Cristalografia por Raios X , Difusão Dinâmica da Luz , Escherichia coli/metabolismo , Técnicas In Vitro , Modelos Moleculares , Mutação , Proteínas Recombinantes , Telomerase/genéticaRESUMO
Strand displacement DNA synthesis (SDDS) is an essential step in DNA replication. With magnetic tweezers, we investigated SDDS kinetics of wild-type gp90 and its exonuclease-deficient polymerase gp90 exo- at single-molecule level. A novel binding state of gp90 to the fork flap was confirmed prior to SDDS, suggesting an intermediate in the initiation of SDDS. The rate and processivity of SDDS by gp90 exo- or wt-gp90 are increased with force and dNTP concentration. The rate and processivity of exonuclease by wt-gp90 are decreased with force. High GC content decreases SDDS and exonuclease processivity but increases exonuclease rate for wt-gp90. The high force and dNTP concentration and low GC content facilitate the successive SDDS but retard the successive exonuclease for wt-gp90. Furthermore, increasing GC content accelerates the transition from SDDS or exonuclease to exonuclease. This work reveals the kinetics of SDDS in detail and offers a broader cognition on the regulation of various factors on SDDS at single-polymerase level.
Assuntos
Bacteriófagos/fisiologia , Replicação do DNA , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Pseudomonas aeruginosa/virologia , Análise de Célula Única/métodos , DNA Polimerase Dirigida por DNA/genética , Recombinação GenéticaRESUMO
Pif1 is an SF1B helicase that is evolutionarily conserved from bacteria to humans and plays multiple roles in maintaining genome stability in both nucleus and mitochondria. Though highly conserved, Pif1 family harbors a large mechanistic diversity. Here, we report crystal structures of Thermus oshimai Pif1 (ToPif1) alone and complexed with partial duplex or single-stranded DNA. In the apo state and in complex with a partial duplex DNA, ToPif1 is monomeric with its domain 2B/loop3 adopting a closed and an open conformation, respectively. When complexed with a single-stranded DNA, ToPif1 forms a stable dimer with domain 2B/loop3 shifting to a more open conformation. Single-molecule and biochemical assays show that domain 2B/loop3 switches repetitively between the closed and open conformations when a ToPif1 monomer unwinds DNA and, in contrast with other typical dimeric SF1A helicases, dimerization has an inhibitory effect on its helicase activity. This mechanism is not general for all Pif1 helicases but illustrates the diversity of regulation mechanisms among different helicases. It also raises the possibility that although dimerization results in activation for SF1A helicases, it may lead to inhibition for some of the other uncharacterized SF1B helicases, an interesting subject warranting further studies.
Assuntos
Proteínas de Bactérias , DNA Helicases , DNA de Cadeia Simples/metabolismo , Thermus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Multimerização ProteicaRESUMO
The candidate anticancer drug curaxins can insert into DNA base pairs and efficiently inhibit the growth of various cancers. However, how curaxins alter the genomic DNA structure and affect the DNA binding property of key proteins remains to be clarified. Here, we first showed that curaxin CBL0137 strongly stabilizes the interaction between the double strands of DNA and reduces DNA bending and twist rigidity simultaneously, by single-molecule magnetic tweezers. More importantly, we found that CBL0137 greatly impairs the binding of CTCF but facilitates trapping FACT on DNA. We revealed that CBL0137 clamps the DNA double helix that may induce a huge barrier for DNA unzipping during replication and transcription and causes the distinct binding response of CTCF and FACT on DNA. Our work provides a novel mechanical insight into CBL0137's anticancer mechanisms at the nucleic acid level.
Assuntos
Carbazóis/farmacologia , DNA/efeitos dos fármacos , Antineoplásicos/farmacologia , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Carbazóis/química , Linhagem Celular , Linhagem Celular Tumoral , DNA/metabolismo , Proteínas de Ligação a DNA , Humanos , Microscopia de Força Atômica/métodos , Pinças Ópticas , Ligação Proteica , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Monoubiquitination at lysine 119 of histone H2A (ubH2A) is a prevalent post-translational modification that is associated with gene repression in the context of chromatin. However, the direct function of ubH2A on nucleosome is poorly understood. Here we identified the effect of ubH2A on nucleosome using single-molecule magnetic tweezers. We revealed that ubH2A stabilizes the nucleosome by blocking the peeling of DNA from the histone octamer. Each ubH2A reinforces one-half of the outer wrap and introduces a robust asymmetry for nucleosome unfolding. Furthermore, a real-time deubiquitination process confirmed that ubH2A-nucleosome is sequentially deubiquitinated and restored to the unmodified nucleosome state. These results provide a novel mechanism to understand the repression of the passage of RNA or DNA polymerases through the ubH2A-nucleosome barrier during gene transcription or replication.
Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitinação , DNA/metabolismo , Histonas/química , Humanos , Lisina/química , Estabilidade Proteica , Ubiquitina Tiolesterase/metabolismoRESUMO
RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3'-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5'-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.
Assuntos
DNA/metabolismo , Escherichia coli/enzimologia , Quadruplex G , RecQ Helicases/metabolismo , Reparo do DNA , Transferência Ressonante de Energia de Fluorescência , Humanos , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a family of plant-specific transcription factors harboring a conserved Lateral Organ Boundaries (LOB) domain, are regulators of plant organ development. Recent studies have unraveled additional pivotal roles of the LBD protein family beyond defining lateral organ boundaries, such as pollen development and nitrogen metabolism. The structural basis for the molecular network of LBD-dependent processes remains to be deciphered. Here, we solved the first structure of the homodimeric LOB domain of Ramosa2 from wheat (TtRa2LD) to 1.9 Å resolution. Our crystal structure reveals structural features shared with other zinc-finger transcriptional factors, as well as some features unique to LBD proteins. Formation of the TtRa2LD homodimer relied on hydrophobic interactions of its coiled-coil motifs. Several specific motifs/domains of the LBD protein were also involved in maintaining its overall conformation. The intricate assembly within and between the monomers determined the precise spatial configuration of the two zinc fingers that recognize palindromic DNA sequences. Biochemical, molecular modeling, and small-angle X-ray scattering experiments indicated that dimerization is important for cooperative DNA binding and discrimination of palindromic DNA through a molecular calipers mechanism. Along with previously published data, this study enables us to establish an atomic-scale mechanistic model for LBD proteins as transcriptional regulators in plants.
Assuntos
Proteínas de Plantas/química , Fatores de Transcrição/química , Triticum/química , Motivos de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Proteínas de Plantas/genética , Domínios Proteicos , Fatores de Transcrição/genética , Triticum/genéticaRESUMO
Intracellular transport of cellular proteins and organelles is critical for establishing and maintaining intracellular organization and cell physiology. Apoptosis is a process of programmed cell death with dramatic changes in cell morphology and organization, during which signaling molecules are transported between different organelles within the cells. However, how the intracellular transport changes in cells undergoing apoptosis remains unknown. Here, we study the dynamics of intracellular transport by using the single-particle tracking method and find that both directed and diffusive motions of endocytic vesicles are accelerated in early apoptotic cells. With careful elimination of other factors involved in the intracellular transport, the reason for the acceleration is attributed to the elevation of adenosine triphosphate (ATP) concentration. More importantly, we show that the accelerated intracellular transport is critical for apoptosis, and apoptosis is delayed when the dynamics of intracellular transport is regulated back to the normal level. Our results demonstrate the important role of transport dynamics in apoptosis and shed light on the apoptosis mechanism from a physical perspective.
Assuntos
Apoptose , Células/metabolismo , Citosol/metabolismo , Células A549 , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Células/citologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , HumanosRESUMO
The G-triplex is a kind of DNA structure formed by G-rich sequences. Previous studies have shown that it is an intermediate for the folding of G-quadruplex and has an antiparallel structure. The folding dynamics of this G-triplex structure, however, have not been well studied until now. In addition, whether a parallel G-triplex structure exists, remains unknown. In this study, by using single-molecule fluorescence resonance energy transfer and circular dichroism spectroscopy methods, we have studied the folding dynamics of the G-triplex and revealed at the single-molecule level, for the first time, that G-triplexes have both parallel and antiparallel structures. Moreover, we have investigated the effects of proximal DNA on G-triplex folding. We have found that both single-stranded TTA and double-stranded DNA at either end of a G-triplex sequence can reduce its folding speed. More interestingly, when located at the 5' end of a G-triplex sequence, the proximal DNA will favor the folding of parallel over antiparallel G-triplex structures. As G-triplex is an intermediate for G-quadruplex folding, the present results may also shed new light on the folding properties of G-quadruplex structures, in terms of dynamics, stability, and the effects of proximal DNA.
Assuntos
DNA/química , Dicroísmo Circular , Transferência Ressonante de Energia de Fluorescência , Conformação de Ácido NucleicoRESUMO
Helicase DHX36 plays essential roles in cell development and differentiation at least partially by resolving G-quadruplex (G4) structures. Here we report crystal structures of the Drosophila homolog of DHX36 (DmDHX36) in complex with RNA and a series of DNAs. By combining structural, small-angle X-ray scattering, molecular dynamics simulation, and single-molecule fluorescence studies, we revealed that positively charged amino acids in RecA2 and OB-like domains constitute an elaborate structural pocket at the nucleic acid entrance, in which negatively charged G4 DNA is tightly bound and partially destabilized. The G4 DNA is then completely unfolded through the 3'-5' translocation activity of the helicase. Furthermore, crystal structures and DNA binding assays show that G-rich DNA is preferentially recognized and in the presence of ATP, specifically bound by DmDHX36, which may cooperatively enhance the G-rich DNA translocation and G4 unfolding. On the basis of these results, a conceptual G4 DNA-resolving mechanism is proposed.
Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , DNA/química , Drosophila/metabolismo , RNA/química , Animais , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Drosophila/química , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Quadruplex G , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Desdobramento de Proteína , RNA/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The Saccharomyces cerevisiae Pif1 protein (ScPif1p) is the prototypical member of the Pif1 family of DNA helicases. ScPif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and suppresses genome instability at G-quadruplex motifs. Here, we report the crystal structures of a truncated ScPif1p (ScPif1p237-780) in complex with different ssDNAs. Our results have revealed that a yeast-specific insertion domain protruding from the 2B domain folds as a bundle bearing an α-helix, α16. The α16 helix regulates the helicase activities of ScPif1p through interactions with the previously identified loop3. Furthermore, a biologically relevant dimeric structure has been identified, which can be further specifically stabilized by G-quadruplex DNA. Basing on structural analyses and mutational studies with DNA binding and unwinding assays, a potential G-quadruplex DNA binding site in ScPif1p monomers is suggested. Our results also show that ScPif1p uses the Q-motif to preferentially hydrolyze ATP, and a G-rich tract is preferentially recognized by more residues, consistent with previous biochemical observations. These findings provide a structural and mechanistic basis for understanding the multifunctional ScPif1p.
Assuntos
Trifosfato de Adenosina/química , DNA Helicases/química , DNA Fúngico/química , DNA de Cadeia Simples/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Quadruplex G , Expressão Gênica , Hidrólise , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TermodinâmicaRESUMO
Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli.