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DNA "breathing" is a thermally driven process in which basepaired DNA sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded DNA coding templates located in the double-stranded DNA "interior," and it is likely that fluctuations of the sugar-phosphate backbones of dsDNA that result in mechanistically useful local base pair opening reactions can be exploited by such DNA regulatory proteins. Such motions are difficult to observe in bulk measurements, both because they are infrequent and because they often occur on microsecond time scales that are not easy to access experimentally. We report single-molecule fluorescence experiments with polarized light, in which tens-of-microseconds rotational motions of internally labeled iCy3/iCy5 donor-acceptor Förster resonance energy transfer fluorophore pairs that have been rigidly inserted into the backbones of replication fork constructs are simultaneously detected using single-molecule Förster resonance energy transfer and single-molecule fluorescence-detected linear dichroism signals. Our results reveal significant local motions in the ∼100-μs range, a reasonable time scale for DNA breathing fluctuations of potential relevance for DNA-protein interactions. Moreover, we show that both the magnitudes and the relaxation times of these backbone breathing fluctuations are significantly perturbed by interactions of the fork construct with a nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting that these motions may play a fundamental role in the initial binding, assembly, and function of the processive helicase-primase (primosome) component of the bacteriophage T4-coded DNA replication complex.smFRET | single-molecule linear dichroism | thermal fluctuations | T4 primosome helicase | DNA helicase D NA "breathing" is defined as the transient opening, due to thermal fluctuations, of nucleic acid base pairs at experimental temperatures below the melting temperature of dsDNA duplex. Such fluctuations are thought to be important to the function of replication, transcription, recombination, and repair systems, which depend on the ability of the relevant protein complexes to gain access to ssDNA templates that are located in the dsDNA "interior" (1-3). Furthermore, the ability of the genome to spontaneously expose partial template sequences is a central feature of protein-DNA binding models in which open conformations serve as substrates that can be "trapped" by a functional protein complex. McConnell and von Hippel (4) suggested that breathing of duplex DNA might be structurally decomposed into a set of distinct breathing elements, each involving characteristic fluctuations of the sugar-phosphate backbone and the nucleobases. For example, a possible motion might involve compression along the double-helix axis (perhaps coupled with twisting or bending), leading to the disruption of WatsonCrick hydrogen bonds without...

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DNA constructs labeled with cyanine fluorescent dyes are important substrates for single-molecule (sm) studies of the functional activity of protein–DNA complexes. We previously studied the local DNA backbone fluctuations of replication fork and primer–template DNA constructs labeled with Cy3/Cy5 donor–acceptor Förster resonance energy transfer (FRET) chromophore pairs and showed that, contrary to dyes linked ‘externally’ to the bases with flexible tethers, direct ‘internal’ (and rigid) insertion of the chromophores into the sugar-phosphate backbones resulted in DNA constructs that could be used to study intrinsic and protein-induced DNA backbone fluctuations by both smFRET and sm Fluorescent Linear Dichroism (smFLD). Here we show that these rigidly inserted Cy3/Cy5 chromophores also exhibit two additional useful properties, showing both high photo-stability and minimal effects on the local thermodynamic stability of the DNA constructs. The increased photo-stability of the internal labels significantly reduces the proportion of false positive smFRET conversion ‘background’ signals, thereby simplifying interpretations of both smFRET and smFLD experiments, while the decreased effects of the internal probes on local thermodynamic stability also make fluctuations sensed by these probes more representative of the unperturbed DNA structure. We suggest that internal probe labeling may be useful in studies of many DNA–protein interaction systems.

The microstructural change related with the mechanical properties of a friction stir welded 6061 Al alloy has been investigated under various welding conditions. Frictional heat and plastic flow during friction stir welding produced fine and equiaxed grains in the stir zone, macroscopically upset and elongated grains in the thermo-mechanically affected zone caused by dynamic recovery and recrystallization. The heat-affected zone, characterized by coarse precipitates, was formed beside the weld zone. Hardness distribution near the weld zone was strongly related to the behavior of precipitates and dislocation density. Especially, hardness of the SZ at a higher tool rotation speed was higher than that of a lower tool rotation speed due to higher density of spherical shaped re-precipitates. The joint strength was approximately 200 MPa which was lower than that of the base metal, 270 MPa, because softening region was formed around the weld zone.