13 research outputs found
Measurement of the salt-dependent stabilization of partially open DNA by Escherichia coli SSB protein
The rezipping force of two complementary DNA strands under tension has been measured in the presence of Escherichia coli single-stranded-binding proteins under salt conditions ranging from 10– to 400 mM NaCl. The effectiveness of the binding protein in preventing rezipping is strongly dependent on salt concentration and compared with the salt dependence in the absence of the protein. At concentrations less than 50 mM NaCl, the protein prevents complete rezipping of λ-phage on the 2-s timescale of the experiment, when the ssDNA is under tensions as low as 3.5 ± 1 pN. For salt concentrations greater than 200 mM NaCl, the protein inhibits rezipping but cannot block rezipping when the tension is reduced below 6 ± 1.8 pN. This change in effectiveness as a function of salt concentration may correspond to salt-dependent changes in binding modes that were previously observed in bulk assays
DNA unzipped under a constant force exhibits multiple metastable intermediates
Single molecule studies, at constant force, of the separation of
double-stranded DNA into two separated single strands may provide information
relevant to the dynamics of DNA replication. At constant applied force, theory
predicts that the unzipped length as a function of time is characterized by
jumps during which the strands separate rapidly, followed by long pauses where
the number of separated base pairs remains constant. Here, we report previously
uncharacterized observations of this striking behavior carried out on a number
of identical single molecules simultaneously. When several single lphage
molecules are subject to the same applied force, the pause positions are
reproducible in each. This reproducibility shows that the positions and
durations of the pauses in unzipping provide a sequence-dependent molecular
fingerprint. For small forces, the DNA remains in a partially unzipped state
for at least several hours. For larger forces, the separation is still
characterized by jumps and pauses, but the double-stranded DNA will completely
unzip in less than 30 min
Comparison of the measured phase diagrams in the force-temperature plane for the unzipping of two different natural DNA sequences
In this work, we consider the critical force required to unzip two different naturally occurring sequences of double-stranded DNA (dsDNA) at temperatures ranging from 20 °
C to 50 °C, where one of the sequences has a 53% average guanine-cytosine (GC) content and the other has a 40% GC content. We demonstrate that the force required to separate the dsDNA of the 53% GC sequence into single-stranded DNA (ssDNA) is approximately 0.5 pN, or approximately 5% greater than the critical force required to unzip the 40% GC sequence at the same temperature. In the temperature range between 20 and 40 °C the measured critical forces correspond reasonably well to predictions based on a simple theoretical homopolymeric model, but at temperatures above 40 °C the measured critical forces are much smaller than the predicted forces. The correspondence between theory and experiment is not improved by using Monte Carlo simulations that consider the heteropolymeric nature of the sequences
Single molecule detection of direct, homologous, DNA/DNA pairing
Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the “default” option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here