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...
Junctions between ssDNA and dsDNA sequences are important in many cellular processes, including DNA replication, transcription, recombination, and repair. Significant transient conformational fluctuations (''DNA breathing'') can occur at these ssDNA-dsDNA junctions. The involvement of such breathing in the mechanisms of macromolecular complexes that operate at these loci is not well understood, in part because these fluctuations have been difficult to measure in a position-specific manner. To address this issue we constructed forked or primer-template DNA constructs with 1 or 2 adjacent 2-aminopurine (2-AP) nucleotide residues (adenine analogues) placed at specific positions on both sides of the ssDNAdsDNA junction. Unlike canonical DNA bases, 2-AP absorbs, fluoresces, and displays CD spectra at wavelengths >300 nm, where other nucleic acid and protein components are transparent. We used CD and fluorescence spectra and acrylamide quenching of these probes to monitor the extent and nature of DNA breathing of A-T base pairs at specific positions around the ssDNA-dsDNA junction. As expected, spectroscopically measurable unwinding penetrates Ϸ2 bp into the duplex region of these junctions under physiological conditions for the constructs examined. Surprisingly, we found that 2-AP bases at ssDNA sites directly adjacent to ssDNA-dsDNA junctions are significantly more unstacked than those at more distant ssDNA positions. These local and transient DNA conformations on both sides of ssDNA-dsDNA junctions may serve as specific interaction targets for enzymes that manipulate DNA in the processes of gene expression.2-aminopurine ͉ circular dichroism ͉ DNA unwinding ͉ fluorescence ͉ forked DNA D ouble-stranded DNA molecules are stabilized by a network of interstrand hydrogen bonds between complementary A-T and G-C base pairs and by intrastrand base stacking. These DNA duplexes exist primarily in the Watson-Crick B-form conformation in aqueous solution at physiological temperature and salt concentration. Heating dsDNA induces a cooperative transition to singlestranded components; the midpoint of this transition is defined as the melting temperature (T m ), which depends on base composition, base sequence, and solvent conditions. dsDNA molecules also experience small thermal ''breathing'' fluctuations that transiently break hydrogen bonds and unstack bases at temperatures well below T m . These fluctuations may play a role in the initial exposure of specific ssDNA binding targets that are otherwise buried in the duplex DNA (promoters, recombination or repair sites, etc.) to various regulatory protein complexes. However, this ''interior DNA breathing'' involves a very small fraction (Ϸ10 Ϫ6 ) of the total DNA at any one time and consequently cannot be observed by normal spectroscopic techniques. Methods such as hydrogen exchange (1-3) or chemical probes that interact irreversibly with and trap ssDNA sequences (for example, formaldehyde or dimethyl sulfate (4) can be used to monitor this interior breathing by accumulating the si...
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 10 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.
Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these ‘macromolecular machines’. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3′-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2–3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3′-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and ‘DNA map’) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.
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