DNA encodes genetic information, which is replicated by DNA polymerase. DNA also serves as a docking station where proteins bind and control processes occurring on the DNA. Here we investigate how DNA is replicated and how DNA-protein interactions are orchestrated. Highly sensitive and parallel single-molecule assays are developed to probe the physical and functional properties of DNA that have been inaccessible with traditional ensemble methods.;We first describe a multiplexed, single-molecule assay based on flow-stretched DNA molecules. We elaborate on our effort to increase the long-term stability of the assay. The assay is characterized as limited to only ∼10 nanometer drift over a time scale of hours and ∼15 nanometer accuracy in determining bead positions. The new development has broadened the application of the flow-stretching assay to studies of various DNA-protein interactions.;Using the flow-stretching assay, we have investigated the sequence-dependent strand displacement mechanism of HIV reverse transcriptase on single-stranded DNA templates. Analysis of the sequence and force-dependent polymerization rate reveals a hybrid mechanism, where the opening of a hairpin is driven by the free energy released during dNTP hydrolysis and the thermal fraying of base-pair interactions.;In contrast to HIV-1 reverse transcriptase, phi29 DNA polymerase exhibits robust primer extension and processive strand displacement activities. We have characterized its DNA polymerization activities, including its processivity during strand displacement synthesis. We have compared the mechano-chemical coupling in several DNA polymerases and deduced the significance of their molecular interactions with the DNA template in modulating replication processes.;As for DNA-protein interactions, DNA has been largely considered a mere template that provides recognition sequences for proteins. However, when multiple proteins bind in a certain region of the genomic DNA, their binding affinity can be altered by the presence of other proteins. We address this question by measuring the binding stability of lac repressor on DNA as a function of its relative distance to the second DNA-protein interaction unit. We have found that the mechanical stress within the DNA double helix yields the long-range interactions between two DNA-binding proteins. The new findings on the allostery in DNA reveal another level of complexity in DNA-protein interactions.
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