Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.

[1]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.

[2]  Aiko Yoshida,et al.  High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events , 2013, Scientific Reports.

[3]  K. Takeyasu,et al.  Motion of the Ca2+‐pump captured , 2011, The FEBS journal.

[4]  D. Lilley,et al.  Structural dynamics of individual Holliday junctions , 2003, Nature Structural Biology.

[5]  Masayuki Endo,et al.  Visualization of dynamic conformational switching of the G-quadruplex in a DNA nanostructure. , 2010, Journal of the American Chemical Society.

[6]  Stephen C. West,et al.  Molecular views of recombination proteins and their control , 2003, Nature Reviews Molecular Cell Biology.

[7]  R. Bennett,et al.  RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. Rafferty,et al.  The structure of Bacillus subtilis RecU Holliday junction resolvase and its role in substrate selection and sequence-specific cleavage. , 2005, Structure.

[9]  D. Leach,et al.  Recombination at double-strand breaks and DNA ends: conserved mechanisms from phage to humans. , 2001, Molecular cell.

[10]  D. Lilley,et al.  Structures of helical junctions in nucleic acids , 2000, Quarterly Reviews of Biophysics.

[11]  Jean-Louis Mergny,et al.  Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame , 2013, Nucleic acids research.

[12]  Yangyang Yang,et al.  Single-molecule visualization of the hybridization and dissociation of photoresponsive oligonucleotides and their reversible switching behavior in a DNA nanostructure. , 2012, Angewandte Chemie.

[13]  Masayuki Endo,et al.  A versatile DNA nanochip for direct analysis of DNA base-excision repair. , 2010, Angewandte Chemie.

[14]  M. Cozar,et al.  Genetic Recombination in Bacillus subtilis 168: Contribution of Holliday Junction Processing Functions in Chromosome Segregation , 2004, Journal of bacteriology.

[15]  S. Kelly,et al.  Structure, flexibility, and mechanism of the Bacillus stearothermophilus RecU holliday junction resolvase , 2007, Proteins.

[16]  Masayuki Endo,et al.  Regulation of DNA methylation using different tensions of double strands constructed in a defined DNA nanostructure. , 2010, Journal of the American Chemical Society.

[17]  D. Lilley Analysis of branched nucleic acid structure using comparative gel electrophoresis , 2008, Quarterly Reviews of Biophysics.

[18]  R. Lurz,et al.  Bacillus subtilis RecU protein cleaves Holliday junctions and anneals single-stranded DNA. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[19]  D. Lilley,et al.  Exchange between stacking conformers in a four-Way DNA junction. , 1998, Biochemistry.

[20]  Hilla Peretz,et al.  Ju n 20 03 Schrödinger ’ s Cat : The rules of engagement , 2003 .

[21]  Taekjip Ha,et al.  Single-molecule four-color FRET. , 2010, Angewandte Chemie.

[22]  M. Nowotny,et al.  Crystal structure of RuvC resolvase in complex with Holliday junction substrate , 2013, Nucleic acids research.

[23]  M. F. White,et al.  The junction-resolving enzymes , 2001, Nature reviews. Molecular cell biology.

[24]  R. G. Lloyd,et al.  Rescue of stalled replication forks by RecG: Simultaneous translocation on the leading and lagging strand templates supports an active DNA unwinding model of fork reversal and Holliday junction formation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[25]  N. Cozzarelli,et al.  Positive Torsional Strain Causes the Formation of a Four-way Junction at Replication Forks* , 2001, The Journal of Biological Chemistry.

[26]  J. Rafferty,et al.  The stalk region of the RecU resolvase is essential for Holliday junction recognition and distortion. , 2011, Journal of molecular biology.