The conformation of DNA molecules electrostatically bound to fluid cationic lipid bilayers is investigated by fluorescence microscopy. The DNA diffuses freely in the plane and follows Rouse dynamics, D ϳ 1͞N, with an increasing number of base pairs N. The chain extension scales as ͗R 2 ͘ ϳ N 2n , with n 0.79 6 0.04 in good agreement with the exact exponent n 3͞4 for a selfavoiding random walk in two dimensions. The structure factor of dilute and semidilute DNA solutions shows fractal scaling behavior, S͑k͒ ϳ k 21͞n. In highly concentrated two-dimensional DNA solutions, the chains were found to segregate. [S0031-9007(99)08573-7]

Force production by type IV pilus retraction is critical for infectivity of Neisseria gonorrhoeae and DNA transfer. We investigated the roles of pilus number and the retraction motor, PilT, in force generation in vivo at the single-molecule level and found that individual retraction events are generated by a single pilus fiber, and only one PilT complex powers retraction. Retraction velocity is constant at low forces but decreases at forces greater than 40 pN, giving a remarkably high average stall force of 110 ± 30 pN. Further insights into the molecular mechanism of force generation are gained from the effect of ATP-depletion, which reduces the rate of retraction but not the stall force. Energetic considerations suggest that more than one ATP is involved in the removal of a single pilin subunit from a pilus. The results are most consistent with a model in which the ATPase PilT forms an oligomer that disassembles the pilus by a cooperative conformational change.

No abstract

A new approach to the study of DNA͞protein interactions has been opened through the recent advances in the manipulation of single DNA molecules. These allow the behavior of individual molecular motors to be studied under load and compared with bulk measurements. One example of such a motor is the DNA polymerase, which replicates DNA. We measured the replication rate by a single enzyme of a stretched single strand of DNA. The marked difference between the elasticity of single-and double-stranded DNA allows for the monitoring of replication in real time. We have found that the rate of replication depends strongly on the stretching force applied to the template. In particular, by varying the load we determined that the biochemical steps limiting replication are coupled to movement. The replication rate increases at low forces, decreases at forces greater than 4 pN, and ceases when the single-stranded DNA substrate is under a load greater than Ϸ20 pN. The decay of the replication rate follows an Arrhenius law and indicates that multiple bases on the template strand are involved in the rate-limiting step of each cycle. This observation is consistent with the induced-fit mechanism for error detection during replication.molecular motors ͉ DNA elasticity D NA polymerases (DNAPs) are responsible for the synthesis of a new DNA strand on a single-stranded (ss) template (1). They play a key role in the replication, repair, and proofreading of DNA by catalyzing the addition of a complementary dNTP to the 3Ј end of the growing strand. The rate of replication of a DNAP, its fidelity, and its processivity (its uninterrupted association with the template) are related to the enzyme's function in vivo.Typically, fast and processive enzymes are associated with quick replication. The simplest example of such an enzyme is T7 DNAP coupled to Escherichia coli thioredoxin (2), which may incorporate several thousand nucleotides at a rate of Ϸ300 bases per second (b͞s) without dissociating from its template (3). A mutant of T7 DNAP (Sequenase), lacking 28 amino acids and the associated exonuclease activity, retains these properties (4) and was studied in this report.Slower, less processive enzymes are usually responsible for repair (and have an auxiliary role in replication). Possessing 5Ј33Ј as well as 3Ј35Ј exonuclease activity, DNAP I from E. coli is representative of this group of DNAPs (1). The 5Ј33Ј exonuclease activity is situated on the smaller fragment of the tri-domain enzyme and may be eliminated by proteolysis. The remaining large (Klenow) fragment, which was also studied here, has a low processivity (1Ϫ100 bases) and a slow replication rate (15 b͞s) (5). As with T7 DNAP, the 3Ј35Ј exonuclease activity of the Klenow fragment may be abolished by a mutation without significantly altering its polymerase activity (6, 7).Although T7 DNAP and E. coli DNAP I have different functions and replication rates, they are structurally similar: they resemble a right hand with the active polymerization site in the palm, the dNTP-binding region...

The elastic properties of single stranded (ss)DNA, studied by pulling on an isolated molecule, are shown to agree with a recent model of ssDNA that takes into account base pairings and screened electrostatic repulsion of the phosphodiester backbone. By an appropriate physicochemical treatment, the pairing interactions were suppressed and ssDNA used as an experimental model for a generic polyelectrolyte. The elastic behavior of such an altered ssDNA deviates strongly from the behavior of an ideal polymer. This deviation is shown to result from the elasticity of the chain and its electrostatic selfavoiding interactions.

The polymeric properties of DNA molecules, which are electrostatically bound to glasssupported cationic lipid membranes, are investigated. The electrostatic interaction is sufficiently strong to hold DNA flat onto the fluid lipid surface but allows DNA to diffuse freely in-plane. The molecules are fluorescently labeled, and fluorescence images are examined in terms of real-space monomer distributions of polymer chains. The chain extension of single DNA fragments of restriction enzyme digests shows power law scaling with number of base pairs in accordance with self-avoiding walks in two dimensions. Dynamic scaling is found for center-of-mass diffusion following Rouse dynamics, D ∼ 1/N, and for rotational relaxation times, τ r ∝ N µ with µ ) 2.6 ( 0.4. A crowded surface of monodisperse λ-DNA behaves like a two-dimensional semidilute solution with a measurable correlation length ξ being smaller than for dilute preparations. Polymer unbinding and the role of surface defects are discussed.

The Gram-positive, rod-forming bacterium Bacillus subtilis efficiently binds and internalizes transforming DNA. The localization of several competence proteins, required for DNA uptake, has been studied using fluorescence microscopy. At least three proteins (ComGA, ComFA, and YwpH) are preferentially associated with the cell poles and appear to colocalize. This association is dynamic; the proteins accumulate at the poles as transformability develops and then delocalize as transformability wanes. DNA binding and uptake also occur preferentially at the cell poles, as shown using fluorescent DNA and in single-molecule experiments with laser tweezers. In addition to the prominent polar sites, the competence proteins also localize as foci in association with the lateral cell membrane, but this distribution does not exhibit the same temporal changes as the polar accumulation. The results suggest the regulated assembly and disassembly of a DNA-uptake machine at the cell poles.

Type IV pili (T4P) are surface structures that undergo extension/retraction oscillations to generate cell motility. In Myxococcus xanthus, T4P are unipolarly localized and undergo pole-to-pole oscillations synchronously with cellular reversals. We investigated the mechanisms underlying these oscillations. We show that several T4P proteins localize symmetrically in clusters at both cell poles between reversals, and these clusters remain stationary during reversals. Conversely, the PilB and PilT motor ATPases that energize extension and retraction, respectively, localize to opposite poles with PilB predominantly at the piliated and PilT predominantly at the non-piliated pole, and these proteins oscillate between the poles during reversals. Therefore, T4P pole-to-pole oscillations involve the disassembly of T4P machinery at one pole and reassembly of this machinery at the opposite pole. Fluorescence recovery after photobleaching experiments showed rapid turnover of YFP–PilT in the polar clusters between reversals. Moreover, PilT displays bursts of accumulation at the piliated pole between reversals. These observations suggest that the spatial separation of PilB and PilT in combination with the noisy PilT accumulation at the piliated pole allow the temporal separation of extension and retraction. This is the first demonstration that the function of a molecular machine depends on disassembly and reassembly of its individual parts.