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..An exciting new opportunity of combining fluorescence methods and biomechanical tools to study the structural changes and functions of enzymes that participate in nucleic acid metabolism has also arisen...

..In this issue, Johnson et al. (2007) shed light on this conundrum using a single molecule approach to dissect the behavior of the T7 DNA helicase...

..The most recent examples are discussed, including the hairpin ribozyme, Holliday junction, G-quadruplex, Rep helicase, reverse transcriptase, and combination with mechanical manipulation...

..We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions...

..Here, we discuss practical considerations for its implementation including experimental apparatus, fluorescent probe selection, surface immobilization, single-molecule FRET analysis schemes, and interpretation...

..Therefore, the closed form of Rep bound to a partial duplex DNA appears to be an inhibited form of the enzyme...

..Although bacterial SSB and eukaryotic RPA have been shown to stimulate DNA synthesis by their cognate DNA polymerases, our findings provide the first in vitro biochemical evidence for the conservation of this property in an archaeon...

..Although the diversity shown by archaeal single-stranded DNA-binding proteins is unparalleled, the presence of their simplest form in many organisms across all domains of life is of greater evolutionary consequence...

..Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates...

..Moreover, engineered mutants of the utilized toxin can potentially be exploited in the future applications...

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..Unexpected phenomena have also been observed from individual molecules suggesting extended or alternative functionality of helicases in vivo...

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..This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution...

..Such repetitive unwinding behavior over a short stretch of duplex may help to keep secondary structures resolved during viral genome replication...

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..In contrast, RPA2 did not interfere with XPD-ssDNA binding but markedly slowed down XPD translocation. Mechanistic models of bypassing DNA-bound proteins by the Rad3 family helicases and their biological implications are discussed...

..The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates...

..This functional integration of two RNA molecular patterns may provide a means to specifically sense and counteract replicating viruses...

..This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes...

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..By combining short timescale (nanoseconds) molecular dynamics and long timescale (milliseconds) stochastic-dynamics descriptions, our study suggests a structure-based mechanism of the ATP-powered unidirectional movement of PcrA helicase...

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..This method provides a biofriendly and simple alternative to surface tethering that is ideal for the study of transient and weakly interacting biological complexes...

..In this chapter we provide a general guide for the construction and execution of single-molecule FRET assays for the study of helicase properties and functionalities...

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..These results strongly support a hand-over-hand model of motility, not an inchworm model...

..This strongly indicates that the observed heterogeneity in dynamic properties does not arise as an artifact of surface attachment, but is intrinsic to the nature of the molecules...

..Our data further suggest that Holliday junction does not spend measurable time with any of the helices unstacked, and that the parallel conformations are not populated to a detectable degree...

..This further supports the notion that electrostatic stabilization of the stacked structures dictates the interconversion rates between different structural forms...

..We identify a set of essential residues coevolutionarily coupled in related helicases that should be involved in the allosteric regulation of these motor proteins...

..Dynamic interplay between structural elements, as we demonstrate for the hairpin ribozyme, may be a general theme for other functional RNA molecules...

..Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven...

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..This supports the proposal for a passive role of RecA-loading machineries in SSB removal...

..We then propose that the selective ability of some helicases to bypass or displace a specific ssDNA binding protein may be important for activation of these enzymes for particular DNA maintenance tasks...

..The persistence length thus estimated ranges from 1.5 nm in 2 M NaCl to 3 nm in 25 mM NaCl...

..Since the device displays clearly distinguishable responses even with a single base pair difference, it may lead to a single molecule sensor of minute sequence differences of a target DNA...

..The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction...

..In this exciting era of transitioning from in vitro to in vivo and in situ conditions, it is anticipated that SM fluorescence methodology will become a common tool of molecular biology...

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..We also show that, in the case of multiple trajectories, analyzing them simultaneously gives better results than averaging over individual analysis results...

..The possibility to place fluorescent labels at nearly any desired position and a sophisticated design of the experiment increases the amount of information that can be extracted in contrast to pure mechanical or fluorescence experiments...

..To clarify these issues, we used a new technique, SHRImP, to measure the interhead distance of nearly rigor myosin VI molecules. Our data show a single peak at 29.3 +/- 0.7 nm, in agreement with the straightforward hand-over-hand model...

..We report that the G-quadruplex can be stabilized without preventing the intrinsic intramolecular dynamics of telomeric DNA...

..This method of mapping two-dimensional reaction landscapes at low forces is readily applicable to other nucleic acid systems and their interactions with proteins and enzymes...

..These intermediates are nonemitting but are still able to quench the fluorescence of the donor at a distance scale shorter than conventional fluorescence resonance energy transfer, suggesting novel biophysical applications...

..This interaction contributes to substrate selectivity and increases the stability of the forked DNA-MCM complex, with possible implications for the MCM unwinding mechanism...

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..The ability to monitor the time course of fusion events between two proteoliposomes should be useful for addressing many important issues in SNARE-mediated membrane fusion...

..In light of these observations we propose a model for hRad52-mediated DNA annealing where ssDNA release and dsDNA zippering are coordinated through successive rearrangement of overlapping nucleoprotein complexes...

..This work represents the first demonstration of real-time biochemical reactions observed via single molecule fluorescence lifetime trajectories of immobilized molecules...

..This in situ assay should be applicable to a large variety of future single-molecule fluorescence studies...

..Despite these interesting observations, further biochemical and genetic analyses are required to gain a deeper understanding of the physiological implications of our findings...

..The results show that magnesium ions stabilize the stacked-X form and destabilize the open and tetrahedral intermediates. Overall, our study provides a detailed dynamic model of the Holliday junction undergoing a conformer transition...

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..Our results suggest that the gel phase membrane is not uniform at the microscopic level but is highly dynamic with the rigidity of local environments constantly changing...

..In addition to bridging the gap in optical resolution, this technique may be useful for biophysical or genomic applications, including the generation of super-high-density maps of single-nucleotide polymorphisms...

..These features may contribute to the unusually fast Pb2+-dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use...

..The algorithm is then applied to the analysis of the binding and dissociation of three RecA monomers on a DNA construct...

..We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software...

..The extremely diverse conformations of the human telomeric DNA may have mechanistic implications for the proteins and drugs that recognize G-rich sequences...

..Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA-RNA interactions...

..Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole-dipole interaction...

..The observed stepwise conformational changes provide a basis to understand abortive RNA synthesis during early stages of initiation and promoter escape during the later stages that allows transition to processive elongation...

..It is likely that the stereochemical environment of the methyl group affects the interaction with metal ions in the center of the junction...

..We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca(2+) and Ca(2+)-sensing fusion effectors in neurotransmitter release...

..We observe significant rates of cleavage and ligation, and a bell-shaped pH dependence for both...

..This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds...

..In all of our studies, bulk-phase biochemical experiments, crystallography, and computational studies through collaboration will help us design and interpret our measurements. ..

..In all of our studies, bulk-phase biochemical experiments, crystallography, and computational studies through collaboration will help us design and interpret our measurements. ..

..In all of our studies, bulk-phase biochemical experiments, crystallography, and computational studies through collaboration will help us design and interpret our measurements. ..

..Furthermore we will use these techniques to make new biological discoveries on the activities of RNA enzymes and helicases. ..

..For example, we will measure both the number of helicase monomers bound to DNA and DNA unwinding to determine if the active form of SF1 helicase is monomer or dimer. ..