Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination

Abstract

Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by ‘snap-locking’ of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

Single-Molecule FRET: A Tool to Characterize DNA Nanostructures

“…In wide-field total internal reflection fluorescence microscopy (TIRFM)-based smFRET, the system of interest is immobilized on a surface, typically on a functionalized quartz surface. With significantly high time resolution offered by modern scientific CMOS cameras, simultaneous observation of hundreds of individual molecules in action for a longer period of time has become possible ( Ray et al, 2021 ). As the distance between the donor and the acceptor fluorophores changes, the fluorescence intensities from them change in an anticorrelated fashion.…”

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

“…Dynamics intrinsic to HJ were analyzed to find cognate sequence and achieve site-specific cleavage [88] Kinetic details of GEN1 dimers decomposed HJ were explained [91] Strand sequence A model of reversible branch migration in mobile 3WJ with trinucleotide repeats was proposed, which may help the treatment of diseases [93] RNA secondary structure Metal cation Na + and K + were proved to facilitate the formation of RNA tetraloop-tetraloop receptor tertiary motif [101] Small molecules in solution…”

“…The experimental results indicated that RuvA bounded to the HJ stably through electrostatic interaction to prevent its conformational dynamics. Recently, a more detailed report [ 88 ] not only focused on the binding properties of RuvC and HJ, but also dissected the behavior of the HJ-RuvC complex to continue cleavage after recognizing the cleavage-active sequence. They discovered that RuvC can achieve site-specific cleavage, which may inspire further research using this mechanism of RuvC.…”

“…They discovered that RuvC can achieve site-specific cleavage, which may inspire further research using this mechanism of RuvC. Similarly, GEN1, a cytoplasmic homologous recombination protein, is considered as one of the key factors in resolving the persistent HJ after the dissolution of the nuclear membrane [ 88 , 89 , 90 ]. Based on previous studies, Sobhy et al [ 91 ] combined smFRET with other techniques to explain the kinetic details of how GEN1 dimers decomposed HJ.…”

Editorial: Single-molecule studies of DNA–protein interactions collection 2021

“…( 8 ) and Sujay et al. ( 9 ) combined smFRET and site-specific labelling of DNA substrates to test length-dependent compaction of telomeres and the dynamics of the E. coli resolvase RuvC during Holliday junction resolution, respectively. In addition to the changes in intensity reporting proximity, smFRET experiments provide a wealth of kinetic information spanning multiple imaging channels.…”