DNA Catenation Reveals the Dynamics of DNA Topology During Replication

Castán, Alicia; Hernández, Pablo; Krimer, Dora B.; Schvartzman, Jorge Bernardo

doi:10.1007/978-1-4939-7459-7_5

Cited by 2 publications

(2 citation statements)

References 13 publications

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“…Analysis programs are available upon request. The potential energy of a specific configuration of the Cre-bound DNA complex is given by [ 1 ] where the vector D contains the 6N displacements between neighboring rigid bodies, 0 D are the mechanical-equilibrium displacements for the linear form of the complex, and K is a k B T•rad -2 , and DNA parameters 0 D , K described above. The equilibrium protein geometry was generated from the crystallographic structure of the Cre-loxP synaptic complex (PDB: 5CRX).…”

Section: Fret-based Recombination-kinetics Assays and Analysismentioning

confidence: 99%

“…Changes in DNA topology, manifested by properties such as supercoiling, knotting, and catenation, are integral to numerous cellular processes including DNA replication, transcription, and recombination. [1][2][3][4][5][6][7] Using circular DNA as a model system to investigate these processes allows associated topological changes to be readily characterized by established biophysical techniques such as gel electrophoresis. 8-10 2 However, making connections between changes in topological parameters and perturbations of canonical DNA geometry is not straightforward for several reasons.…”

Section: Introductionmentioning

confidence: 99%

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Loop-closure Kinetics Reveal a Stable, Right-handed DNA Intermediate in Cre Recombination

Shoura

Giovan

Vetcher

et al. 2019

Preprint

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In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of DNA target sites. The strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of the DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. Here we use the kinetics of loop closure involving closely spaced (131-151 bp), directly repeated loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-ofplane HJ distortion. This result unequivocally stands in contrast to the square-planar intermediate geometry determined from crystallographic data for the Cre-loxP system and other int-superfamily recombinases. J measurements carried out with an isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex may result from the temporal averaging of diverse right-handed and achiral structures. Applied to Cre recombinase, and other biological systems, our approach bridges the static pictures provided by crystal structures and the natural dynamics of macromolecules in vivo. This approach thus advances a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.

show abstract

Section: Fret-based Recombination-kinetics Assays and Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Loop-closure Kinetics Reveal a Stable, Right-handed DNA Intermediate in Cre Recombination

Shoura

Giovan

Vetcher

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination

Shoura

Giovan

Vetcher

et al. 2020

Nucleic Acids Research

View full text Add to dashboard Cite

In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of loxP DNA target sites. The enzyme system's strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral, planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. We use the kinetics of loop closure involving closely spaced (131–151 bp) loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-of-plane HJ distortion, which unequivocally stands in contrast to the square-planar intermediate geometry from Cre-loxP crystal structures and those of other int-superfamily recombinases. J measurements for an HJ-isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex is consistent with temporal averaging of right-handed and achiral structures. Our approach connects the static pictures provided by crystal structures and the natural dynamics of macromolecules in solution, thus advancing a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.

show abstract

DNA Catenation Reveals the Dynamics of DNA Topology During Replication

Cited by 2 publications

References 13 publications

Loop-closure Kinetics Reveal a Stable, Right-handed DNA Intermediate in Cre Recombination

Loop-closure Kinetics Reveal a Stable, Right-handed DNA Intermediate in Cre Recombination

Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination

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