DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome.
Self-propelled particles (SPP) exhibit complex collective motions, mimicking autonomous behaviors that are often seen in the natural world, but essentially are generated by simple mutual interactions. Previous research on SPP systems focuses on collective behaviors of a uniform population. However, very little is known about the evolution of individual particles under the same global influence. Here we show self-organized rotating spiral coils in a two-dimensional (2D) active system. By using swarming bacteria Vibrio alginolyticus as an ideal experimental realization of a well-controlled 2D self-propelled system, we study the interaction between ultra-long cells and short background active cells. The self-propulsion of long cells and their interactions with neighboring short cells leads to a self-organized, stable spiral rotational state in 2D. We find four types of spiral coils with two main features: the rotating direction (clockwise or counter-clockwise) and the central structure (single or double spiral). The body length of the spiral coils falls between 32 and 296 μm and their rotational speed is within a range from 2.22 to 22.96 rad s(-1). The dynamics of these spiral coils involves folding and unfolding processes, which require local velocity changes of the long bacterium. This phenomenon can be qualitatively replicated by a Brownian dynamics simulation using a simple rule of the propulsion thrust, imitating the reorientation of bacterial flagella. Apart from the physical and biological interests in swarming cells, the formation of self-organized spiral coils could be useful for the next generation of microfabrication.
Sso10a proteins are small DNA-binding proteins expressed by the crenarchaeal model organism Sulfolobus solfataricus. Based on the structure of Sso10a1, which contains a winged helix-turn-helix motif, it is believed that Sso10a proteins function as sequence-specific transcription factors. Here we show that Sso10a1 and Sso10a2 exhibit different distinct DNA-binding modes. While the ability to bend DNA is shared between the two proteins, DNA bridging is observed only for Sso10a1 and only Sso10a2 exhibits filament formation along DNA. The architectural properties of Sso10a proteins suggest that these proteins fulfil generic roles in chromatin organization and compaction. As these proteins exhibit different binding behaviour depending on their DNA binding stoichiometry, altered levels of expression in the cell can be exploited to drive changes in local genome folding, which may operate to modulate transcription.
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