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.
Ramreddy
T
T
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
Sen
Subhojit
S
Rao
Basuthkar J
BJ
Krishnamoorthy
G
G
D000255:Adenosine Triphosphate / Q000737:chemistry*; D020029:Base Pairing; D004269:DNA, Bacterial / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005454:Fluorescence Polarization; D006868:Hydrolysis; D008027:Light; D009698:Nucleoproteins / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D011938:Rec A Recombinases / Q000737:chemistry*; D011995:Recombination, Genetic; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009698:Nucleoproteins; D009841:Oligonucleotides; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases