9 research outputs found
Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination
Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca2+ than of Mg2+, we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca2+ induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg2+, ADP/Mg2+ or ADP/Ca2+ does not. A high strand exchange activity is observed for the filament formed with ATP/Ca2+, whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca2+ stabilizes the loop conformation and thereby the protein–DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange
UV Transition Moments of Tyrosine
To
assist polarized-light spectroscopy for protein-structure analysis,
the UV spectrum of <i>p</i>-cresol, the chromophore of tyrosine,
was studied with respect to transition moment directions and perturbation
by solvent environment. From linear dichroism (LD) spectra of <i>p</i>-cresol aligned in stretched matrices of poly(vinyl alcohol)
and polyethylene, the lowest π–π* transition (L<sub>b</sub>) is found to have pure polarization over its entire absorption
(250–300 nm) with a transition moment perpendicular to the
symmetry axis (C<sub>1</sub>–C<sub>4</sub>), both in polar
and nonpolar environments. For the second transition (L<sub>a</sub>), polarized parallel with the symmetry axis, a certain admixture
of intensity with orthogonal polarization is noticed, depending on
the environment. While the L<sub>b</sub> spectrum in cyclohexane shows
a pronounced vibrational structure, it is blurred in methanol, which
can be modeled as due to many microscopic polar environments. With
the use of quantum mechanical (QM) calculations, the transition moments
and solvent effects were analyzed with the B3LYP and ωB97X-D
functionals in cyclohexane, water, and methanol using a combination
of implicit and explicit solvent models. The blurred L<sub>b</sub> band is explained by solvent hydrogen bonds, where both accepting
and donating a hydrogen causes energy shifts. The inhomogeneous solvent-shift
sensitivity in combination with robust polarization can be exploited
for analyzing tyrosine orientation distributions in protein complexes
using LD spectroscopy
Minor-Groove Binding Drugs: Where Is the Second Hoechst 33258 Molecule?
Hoechst 33258 binds with high affinity
into the minor groove of
AT-rich sequences of double-helical DNA. Despite extensive studies
of this and analogous DNA binding molecules, there still remains uncertainty
concerning the interactions when multiple ligand molecules are accommodated
within close distance. Albeit not of direct concern for most biomedical
applications, which are at low drug concentrations, interaction studies
for higher drug binding are important as they can give fundamental
insight into binding mechanisms and specificity, including drug self-stacking
interactions that can provide base-sequence specificity. Using circular
dichroism (CD), isothermal titration calorimetry (ITC), and proton
nuclear magnetic resonance (<sup>1</sup>H NMR), we examine the binding
of Hoechst 33258 to three oligonucleotide duplexes containing AT regions
of different lengths: [d(CGCGAATTCGCG)]<sub>2</sub> (A<sub>2</sub>T<sub>2</sub>), [d(CGCAAATTTGCG)]<sub>2</sub> (A<sub>3</sub>T<sub>3</sub>), and [d(CGAAAATTTTCG)]<sub>2</sub> (A<sub>4</sub>T<sub>4</sub>). We find similar binding geometries in the minor groove for all
oligonucleotides when the ligand-to-duplex ratio is less than 1:1.
At higher ratios, a second ligand can be accommodated in the minor
groove of A<sub>4</sub>T<sub>4</sub> but not A<sub>2</sub>T<sub>2</sub> or A<sub>3</sub>T<sub>3</sub>. We conclude that the binding of the
second Hoechst to A<sub>4</sub>T<sub>4</sub> is not cooperative and
that the molecules are sitting with a small separation apart, one
after the other, and not in a sandwich structure as previously proposed
Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament
The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction
Sensing Conformational Changes in DNA upon Ligand Binding Using QCM-D. Polyamine Condensation and Rad51 Extension of DNA Layers
Biosensors,
in which binding of ligands is detected through changes in the optical
or electrochemical properties of a DNA layer confined to the sensor
surface, are important tools for investigating DNA interactions. Here,
we investigate if conformational changes induced in surface-attached
DNA molecules upon ligand binding can be monitored by the quartz crystal
microbalance with dissipation (QCM-D) technique. DNA duplexes
containing 59–184 base pairs were formed on QCM-D crystals
by stepwise assembly of synthetic oligonucleotides of designed base
sequences. The DNA films were exposed to the cationic polyamines spermidine
and spermine, known to condense DNA molecules in bulk experiments,
or to the recombination protein Rad51, known to extend the DNA helix.
The binding and dissociation of the ligands to the DNA films were
monitored in real time by measurements of the shifts in resonance
frequency (Δ<i>f</i>) and in dissipation (Δ<i>D</i>). The QCM-D data were analyzed using a Voigt-based model
for the viscoelastic properties of polymer films in order to evaluate
how the ligands affect thickness and shear viscosity of the DNA layer.
Binding of spermine shrinks all DNA layers and increases their viscosity
in a reversible fashion, and so does spermidine, but to a smaller
extent, in agreement with its lower positive charge. SPR was used
to measure the amount of bound polyamines, and when combined with
QCM-D, the data indicate that the layer condensation leads to a small
release of water from the highly hydrated DNA films. The binding of
Rad51 increases the effective layer thickness of a 59bp film, more
than expected from the know 50% DNA helix extension. The combined
results provide guidelines for a QCM-D biosensor based on ligand-induced
structural changes in DNA films. The QCM-D approach provides high
discrimination between ligands affecting the thickness and the structural
properties of the DNA layer differently. The reversibility of the
film deformation allows comparative studies of two or more analytes
using the same DNA layer as demonstrated here by spermine and spermidine
Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament
The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction
Adjuvant docetaxel, doxorubicin, and cyclophosphamide in node-positive breast cancer: 10-year follow-up of the phase 3 randomised BCIRG 001 trial
We compared standard adjuvant anthracycline chemotherapy with anthracycline-taxane combination chemotherapy in women with operable node-positive breast cancer. Here we report the final, 10-year follow-up analysis of disease-free survival, overall survival, and long-term safety.
BCIRG 001 was an open label, phase 3, multicentre trial in which 1491 patients aged 18-70 years with node-positive, early breast cancer and a Karnofsky score of 80% or more were randomly assigned to adjuvant treatment with docetaxel, doxorubicin, and cyclophosphamide (TAC) or fluorouracil, doxorubicin, and cyclophosphamide (FAC) every 3 weeks for six cycles. Randomisation was stratified according to institution and number of involved axillary lymph nodes per patient (one to three vs four or more). Disease-free survival was the primary endpoint and was defined as the interval between randomisation and breast cancer relapse, second primary cancer, or death, whichever occurred first. Efficacy analyses were based on the intention-to-treat principle. BCIRG 001 is registered with ClinicalTrials.gov, number NCT00688740.
Enrolement took place between June 11, 1997 and June 3, 1999; 745 patients were assigned to receive TAC and 746 patients were assigned to receive FAC. After a median follow-up of 124 months (IQR 90-126), disease-free survival was 62% (95% CI 58-65) for patients in the TAC group and 55% (51-59) for patients in the FAC group (hazard ratio [HR] 0·80, 95% CI 0·68-0·93; log-rank p=0·0043). 10-year overall survival was 76% (95% CI 72-79) for patients in the TAC group and 69% (65-72) for patients in the FAC group (HR 0·74, 0·61-0·90; log-rank p=0·0020). TAC improved disease-free survival relative to FAC irrespective of nodal, hormone receptor, and HER2 status, although not all differences were significant in these subgroup analyses. Grade 3-4 heart failure occurred in 26 (3%) patients in the TAC group and 17 (2%) patients in the FAC group, and caused death in two patients in the TAC group and four patients in the FAC group. A substantial decrease in left ventricular ejection fraction (defined as a relative decrease from baseline of 20% or more) was seen in 58 (17%) patients who received TAC and 41 (15%) patients who received FAC. Six patients who received TAC developed leukaemia or myelodysplasia, as did three patients who received FAC.
Our results provide evidence that the initial therapeutic outcomes seen at the 5-year follow-up with a docetaxel-containing adjuvant regimen are maintained at 10 years. However, a substantial percentage of patients had a decrease in left ventricular ejection fraction, probably caused by anthracycline therapy, which warrants further investigation.
Sanofi