G-quadruplex conformation and dynamics are determined by loop length and sequence

The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that both the loop (non-G components) length and sequence contribute to the conformation of the GQ. Real time single molecule traces reveal that the folding dynamics also depend on the loop composition. We demonstrate that GQ-stabilizing small molecules, N-methyl mesoporphyrin IX (NMM), its analog, NMP and the G4R1 protein bind selectively to the parallel GQ conformation. Our findings point to the complexity of GQ folding governed by the loop length and sequence and how the GQ conformation determines the small molecule and protein binding propensity.     

Real-time detection of cruciform extrusion by single-molecule DNA nanomanipulation

During cruciform extrusion, a DNA inverted repeat unwinds and forms a four-way junction in which two of the branches consist of hairpin structures obtained by self-pairing of the inverted repeats. Here, we use single-molecule DNA nanomanipulation to monitor in real-time cruciform extrusion and rewinding. This allows us to determine the size of the cruciform to nearly base pair accuracy and its kinetics with second-scale time resolution. We present data obtained with two different inverted repeats, one perfect and one imperfect, and extend single-molecule force spectroscopy to measure the torque dependence of cruciform extrusion and rewinding kinetics. Using mutational analysis and a simple two-state model, we find that in the transition state intermediate only the B-DNA located between the inverted repeats (and corresponding to the unpaired apical loop) is unwound, implying that initial stabilization of the four-way (or Holliday) junction is rate-limiting. We thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop, while rewinding is kinetically regulated by features of the stem. These results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and B-form states.     

DNA dynamics in RecA-DNA filaments: ATP hydrolysis-related flexibility in DNA

RecA-catalyzed DNA recombination is initiated by a mandatory, high-energy form of DNA in RecA-nucleoprotein filaments, where bases are highly unstacked and the backbone is highly unwound. Interestingly, only the energetics consequent to adenosine triphosphate (ATP) binding, rather than its hydrolysis, seems sufficient to mediate such a high-energy structural hallmark of a recombination filament. The structural consequence of ATP hydrolysis on the DNA part of the filament thus remains largely unknown. We report time-resolved fluorescence dynamics of bases in RecA-DNA complexes and demonstrate that DNA bases in the same exhibit novel, motional dynamics with a rotational correlation time of 7-10 ns, specifically in the presence of ATP hydrolysis. When the ongoing ATP hydrolysis of RecA-DNA filament is "poisoned" by a nonhydrolyzable form of ATP (ATPgammaS), the motional dynamics cease and reveal a global motion with a rotational correlation time of >20 ns. Such ATP hydrolysis-induced flexibility ensues in single-stranded as well as double-stranded bases of RecA-DNA filaments. These results suggest that the role of ATP hydrolysis is to induce a high level of backbone flexibility in RecA-DNA filament, a dynamic property that is likely to be important for efficient strand exchanges in ATP hydrolysis specific RecA reactions. It is the absence of these motions that may cause high rigidity in RecA-DNA filaments in ATPgammaS. Dynamic light scattering measurement comparisons of RecA-ss-DNA filaments formed in ATPgammaS vs that of ATP confirmed such an interpretation, where the former showed a complex of larger (30 nm) hydrodynamic radius than that of latter (12-15 nm). Taken together, these results reveal a more dynamic state of DNA in RecA-DNA filament that is hydrolyzing ATP, which encourage us to model the role of ATP hydrolysis in RecA-mediated DNA transactions.