15 research outputs found
A Single-Molecule View of the Assembly Pathway, Subunit Stoichiometry, and Unwinding Activity of the Bacteriophage T4 Primosome (helicase–primase) Complex
Single-molecule fluorescence resonance
energy transfer (smFRET)
methods were used to study the assembly pathway and DNA unwinding
activity of the bacteriophage T4 helicase–primase (primosome)
complex. The helicase substrates used were surface-immobilized model
DNA replication forks “internally” labeled in the duplex
region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in
the lagging and leading strands. The time dependence of the smFRET
signals was monitored during the unwinding process, and helicase rates
and processivities were measured as a function of GTP concentration.
This smFRET approach was also used to investigate the subunit stoichiometry
of the primosome and the assembly pathway required to form functional
and fully active primosome–DNA complexes. We confirmed that
gp41 helicase monomer subunits form stable hexameric helicases in
the presence of GTP and that the resulting (gp41)<sub>6</sub> complexes
bind only weakly at DNA fork junctions. The addition of a single subunit
of gp61 primase stabilized the resulting primosome complex at the
fork and resulted in fully active and processive primosome helicases
with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit
ratios substantially reduced the primosome unwinding activity. The
use of alternative assembly pathways resulted in a loss of helicase
activity and the formation of metastable DNA–protein aggregates,
which were easily detected in our smFRET experiments as intense light-scattering
foci. These single-molecule experiments provide a detailed real-time
visualization of the assembly pathway and duplex DNA unwinding activity
of the T4 primosome and are consistent with more indirect equilibrium
and steady state results obtained in bulk solution studies
Sequence-Dependent Conformational Heterogeneity and Proton-Transfer Reactivity of the Fluorescent Guanine Analogue 6‑Methyl Isoxanthopterin (6-MI) in DNA
The
local conformations of individual nucleic acid bases in DNA
are important components in processes fundamental to gene regulation.
Fluorescent nucleic acid base analogues, which can be substituted
for natural bases in DNA, can serve as useful spectroscopic probes
of average local base conformation and conformational heterogeneity.
Here we report excitation–emission peak shift (EES) measurements
of the fluorescent guanine (G) analogue 6-methyl isoxanthoptherin
(6-MI), both as a ribonucleotide monophosphate (NMP) in solution and
as a site-specific substituent for G in various DNA constructs. Changes
in the peak positions of the fluorescence spectra as a function of
excitation energy indicate that distinct subpopulations of conformational
states exist in these samples on time scales longer than the fluorescence
lifetime. Our pH-dependent measurements of the 6-MI NMP in solution
show that these states can be identified as protonated and deprotonated
forms of the 6-MI fluorescent probe. We implement a simple two-state
model, which includes four vibrationally coupled electronic levels
to estimate the free energy change, the free energy of activation,
and the equilibrium constant for the proton transfer reaction. These
parameters vary in single-stranded and duplex DNA constructs, and
also depend on the sequence context of flanking bases. Our results
suggest that proton transfer in 6-MI-substituted DNA constructs is
coupled to conformational heterogeneity of the probe base, and can
be interpreted to suggest that Watson–Crick base pairing between
6-MI and its complementary cytosine in duplex DNA involves a “low-barrier-hydrogen-bond”.
These findings may be important in using the 6-MI probe to understand
local base conformational fluctuations, which likely play a central
role in protein–DNA and ligand–DNA interactions
Examples for the determination of radial magnification errors.
<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p
Analysis of the rotor temperature.
<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p
Absence of a long-term trend in <i>s</i><sub><i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i></sub>-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).
<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p
Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.
<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p
Histogram and box-and-whisker plot of <i>s</i>-values of the BSA monomer after different corrections: Raw experimental <i>s</i>-values (black, with grey histogram), scan time corrected <i>s</i><sub><i>t</i></sub>-values (blue), rotor temperature corrected <i>s</i><sub><i>20T</i></sub>-values (green), or radial magnification corrected <i>s</i><sub><i>r</i></sub>-values (cyan), and fully corrected <i>s</i><sub><i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i></sub>-values (red with red histogram).
<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as a vertical line.</p
Distributions of calculated BSA monomer signals for the different kits and the different optical systems.
<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as vertical line.</p
Correlations of the <i>s</i><sub><i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i></sub>-values of the BSA monomer with the difference of the best-fit meniscus from the mean meniscus value, separately for absorbance data sets (A) and interference data sets (B).
<p>The difference of the best-fit meniscus to the mean was calculated separately for each kit, to eliminate offsets due to different sample volumes in each kit, and then merged into groups for the optical systems. Data are shown as a histogram with frequency values indicated in the colorbar. The dotted lines show the theoretically expected dependence of the apparent <i>s</i>-value on errors in the absolute radial position.</p
Root-mean-square deviation of the best-fit <i>c</i>(<i>s</i>) model of the BSA sedimentation experiment when scanned with the absorbance system (green) and the interference system (magenta).
<p>The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p