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.

HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl2 conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU–DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (w/v), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.

Architectural DNA–binding proteins are involved in many important DNA transactions by virtue of their ability to change DNA conformation. Histone-like protein from E. coli strain U93, HU, is one of the most studied bacterial architectural DNA–binding proteins. Nevertheless, there is still a limited understanding of how the interactions between HU and DNA are affected by ionic conditions and the structure of DNA. Here, using optical tweezers in combination with fluorescent confocal imaging, we investigated how ionic conditions affect the interaction between HU and DNA. We directly visualized the binding and the diffusion of fluorescently labelled HU dimers on DNA. HU binds with high affinity and exhibits low mobility on the DNA in the absence of Mg2+; it moves 30-times faster and stays shorter on the DNA with 8 mM Mg2+ in solution. Additionally, we investigated the effect of DNA tension on HU–DNA complexes. On the one hand, our studies show that binding of HU enhances DNA helix stability. On the other hand, we note that the binding affinity of HU for DNA in the presence of Mg2+ increases at tensions above 50 pN, which we attribute to force-induced structural changes in the DNA. The observation that HU diffuses faster along DNA in presence of Mg2+ compared to without Mg2+ suggests that the free energy barrier for rotational diffusion along DNA is reduced, which can be interpreted in terms of reduced electrostatic interaction between HU and DNA, possibly coinciding with reduced DNA bending.

Acoustic Force Spectroscopy (AFS) is a single-molecule micromanipulation technique that uses sound waves to exert force on surface-tethered DNA molecules in a microfluidic chamber. As large numbers of individual protein-DNA complexes are tracked in parallel, AFS provides insight into the individual properties of such complexes as well as their population averages. In this chapter, we describe in detail how to perform AFS experiments specifically on bare DNA, protein-DNA complexes, and how to extract their (effective) persistence length and contour length from force-extension relations.

Key Word Index-Eenzyl halides; halide ions; Finkelstein reactions.The kinetics of the FinkeIstein reactions of benzyl halides and halide ions in dry acetone was studied by using the GC and HPLC methods. The method of conductivity is used to measure the degree of dissociation of alkali halide in acetone. The dissociation of the salt and the common-ion effect were used to correct the halide ion concentration, Let K,,and K,,+ represent the corrected second-order rate cansfant and equilibrium constant for the PhCH,X-X'reaction, respectively, then a t 2 5 O kc,l--1.83X10-3 M-Is-1, &-,l-=4.48x104; R B ,~~-= O .~I ? M -~ s-l, 24.5"C) were identified by 'H NMR, IR, and GC/MS. Both NMR and IR data showed that the products contained a negligible amount of water. The mass spectrum of benzyI iodide gave m/e (%> (at 70eV): 91 (loo), 65 (lo), 92 (7), 89 (4), 218 (3.25), 127 (as), 77 (0.1), InstrumentsThe following instruments were used in t h i s work: (1) Shimadzu GC-4C with Hewlett Packard 3390 integrator, (2) Varian GC-3300 with Varian SP-4290 integrator, (3j Hewlett Parkard 1084B LC with 79850B LC terminal, (4) Hitachi 260-30 IR spectrometer, (5) Hitachi M-52 Mass spectrometer, (6) Bruker WP lOOFT NMR spectrometer, (7) Tokyo Toa Omnimeter model OM-l A, Japan, (8) Yokogawa YEW 2575 Digital Thermometer, Japan, (9) Colora Thermostat WK-5, (10) Mel-Temperature, Laboratory Devices. ProceduresMeasured volumes of reactant solutions of known concentrations were mixed under stirring to start a kinetic run (e,g., 5 m L ) 0.44 !I11 PhCH,Cl added to 25 nzL 0.088rl.l NaI). The mixing time was within 5 secoizds.

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