DNA Sequencing Using Polymerase Substrate Binding Kinetics
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Similar Datasets
Project description:Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications.
| S-EPMC4354037 | biostudies-literature
Project description:Polymerase II binding analysis during nutrient restriction using targeted DamID
| PRJNA605807 | ENA
Project description:Replicative DNA polymerases use a complex, multistep mechanism for efficient and accurate DNA replication as uncovered by intense kinetic and structural studies. Recently, single-molecule fluorescence spectroscopy has provided new insights into real time conformational dynamics utilized by DNA polymerases during substrate binding and nucleotide incorporation. We have used single-molecule Förster resonance energy transfer techniques to investigate the kinetics and conformational dynamics of Sulfolobus solfataricus DNA polymerase B1 (PolB1) during DNA and nucleotide binding. Our experiments demonstrate that this replicative polymerase can bind to DNA in at least three conformations, corresponding to an open and closed conformation of the finger domain as well as a conformation with the DNA substrate bound to the exonuclease active site of PolB1. Additionally, our results show that PolB1 can transition between these conformations without dissociating from a primer-template DNA substrate. Furthermore, we show that the closed conformation is promoted by a matched incoming dNTP but not by a mismatched dNTP and that mismatches at the primer-template terminus lead to an increase in the binding of the DNA to the exonuclease site. Our analysis has also revealed new details of the biphasic dissociation kinetics of the polymerase-DNA binary complex. Notably, comparison of the results obtained in this study with PolB1 with those from similar single-molecule studies with an A-family DNA polymerase suggests mechanistic differences between these polymerases. In summary, our findings provide novel mechanistic insights into protein conformational dynamics and substrate binding kinetics of a high fidelity B-family DNA polymerase.
| S-EPMC3630881 | biostudies-literature
Project description:Phase separation of RNA-binding protein promotes polymerase binding and transcription
| PRJNA631979 | ENA
Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
2024-05-29 | GSE246484 | GEO
Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
2024-05-29 | GSE246483 | GEO
Project description:Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through inhibition of pause release, we determine that pause-release rates are highly variable and most promoter-proximal paused RNA Polymerase II molecules prematurely terminate (~80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with induction of promoter-proximal termination under hyperosmotic stress but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.
2021-09-13 | GSE166202 | GEO
Project description:Effect of varying RNA concentration amounts and hybridization times on array quality and binding kinetics.
2004-03-23 | E-MEXP-40 | biostudies-arrayexpress
Project description:Enzyme kinetic analysis reveals a dynamic relationship between enzymes and their substrates. Overall enzyme activity can be controlled by both protein expression and various cellular regulatory systems. Interestingly, the availability and concentrations of intracellular substrates can constantly change, depending on conditions and cell types. Here, we review previously reported enzyme kinetic parameters of cellular and viral DNA and RNA polymerases with respect to cellular levels of their nucleotide substrates. This broad perspective exposes a remarkable co-evolution scenario of DNA polymerase enzyme kinetics with dNTP levels that can vastly change, depending on cell proliferation profiles. Similarly, RNA polymerases display much higher Km values than DNA polymerases, possibly due to millimolar range rNTP concentrations found in cells (compared with micromolar range dNTP levels). Polymerases are commonly targeted by nucleotide analog inhibitors for the treatments of various human diseases, such as cancers and viral pathogens. Because these inhibitors compete against natural cellular nucleotides, the efficacy of each inhibitor can be affected by varying cellular nucleotide levels in their target cells. Overall, both kinetic discrepancy between DNA and RNA polymerases and cellular concentration discrepancy between dNTPs and rNTPs present pharmacological and mechanistic considerations for therapeutic discovery.
| S-EPMC7521635 | biostudies-literature
Project description:An RNA-involved phase-separation model has been proposed for transcription control. Yet, the molecular links that connect RNA to the transcription machinery remain missing. Here we find RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), and some are colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs show that the paraspeckle protein PSPC1 inhibits the RNA-induced premature release of Pol II, and makes use of RNA as multivalent molecules to enhance the formation of transcription condensates and subsequent phosphorylation and release of Pol II. This synergistic interplay enhances polymerase engagement and activity via the RNA-binding and phase-separation activities of PSPC1. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II binding and nascent transcription. We propose that promoter-associated RNAs and their binding proteins synergize the phase separation of polymerase condensates to promote active transcription.
2020-05-12 | GSE150399 | GEO