Single molecule detection of direct, homologous, DNA/DNA pairing

Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the “default” option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.

[1]  M. Prentiss,et al.  DNA unzipped under a constant force exhibits multiple metastable intermediates , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Geoff S Baldwin,et al.  DNA double helices recognize mutual sequence homology in a protein free environment. , 2008, The journal of physical chemistry. B.

[3]  Guillaume J. Filion,et al.  Sensing X Chromosome Pairs Before X Inactivation via a Novel X-Pairing Region of the Xic , 2007, Science.

[4]  S. Keeney,et al.  Progression of meiotic DNA replication is modulated by interchromosomal interaction proteins, negatively by Spo11p and positively by Rec8p. , 2000, Genes & development.

[5]  William M. Gelbart,et al.  Counterion-Induced Attraction between Rigid Polyelectrolytes , 1997 .

[6]  B. Shklovskii,et al.  Screening of a macroion by multivalent ions: correlation-induced inversion of charge. , 1999, Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics.

[7]  R. Dickerson,et al.  1 A crystal structures of B-DNA reveal sequence-specific binding and groove-specific bending of DNA by magnesium and calcium. , 2000, Journal of molecular biology.

[8]  J. Dekker,et al.  Capturing Chromosome Conformation , 2002, Science.

[9]  E. Selker.  Gene silencing: repeats that count. , 1999 .

[10]  J. H. Wilson.  Nick-free formation of reciprocal heteroduplexes: a simple solution to the topological problem. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[11]  D. Thirumalai,et al.  Dynamics of tRNA at different levels of hydration. , 2009, Biophysical journal.

[12]  W. Gilbert,et al.  Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis , 1988, Nature.

[13]  K. Yoshikawa,et al.  Na+ shows a markedly higher potential than K+ in DNA compaction in a crowded environment. , 2005, Biophysical journal.

[14]  M. Terasaki,et al.  Evidence that intracellular magnesium is present in cells at a regulatory concentration for protein synthesis. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[15]  A. Kornyshev,et al.  The homology recognition well as an innate property of DNA structure , 2009, Proceedings of the National Academy of Sciences.

[16]  Tarun Jain,et al.  The role of water in protein-DNA recognition. , 2004, Annual review of biophysics and biomolecular structure.

[17]  V. Adrian Parsegian,et al.  DNA‐Inspired Electrostatics , 2000 .

[18]  J. LaSalle,et al.  Homologous Association of Oppositely Imprinted Chromosomal Domains , 1996, Science.

[19]  A. Kornyshev,et al.  Nonlinear effects in the torsional adjustment of interacting DNA. , 2004, Physical review. E, Statistical, nonlinear, and soft matter physics.

[20]  N. Sugimoto,et al.  Nucleic acid duplex stability: influence of base composition on cation effects. , 1999, Nucleic acids research.

[21]  A. Travers,et al.  Self-assembly of double-stranded DNA molecules at nanomolar concentrations. , 2007, Biochemistry.

[22]  J. Haber,et al.  Mechanisms of Rad52-Independent Spontaneous and UV-Induced Mitotic Recombination in Saccharomyces cerevisiae , 2008, Genetics.

[23]  A. Kornyshev,et al.  Sequence recognition in the pairing of DNA duplexes. , 2001, Physical review letters.

[24]  S. Kowalczykowski.  Structural biology: Snapshots of DNA repair , 2008, Nature.

[25]  Abby Dernburg,et al.  Homologous Chromosome Pairing in Drosophila melanogaster Proceeds through Multiple Independent Initiations , 1998, The Journal of cell biology.

[26]  Andrey G. Cherstvy,et al.  Torsional deformation of double helix in interaction and aggregation of DNA. , 2004, The journal of physical chemistry. B.

[27]  N. Hud,et al.  Toroidal DNA condensates: unraveling the fine structure and the role of nucleation in determining size. , 2005, Annual review of biophysics and biomolecular structure.

[28]  N. Kleckner,et al.  Somatic pairing of homologs in budding yeast: existence and modulation. , 1999, Genes & development.

[29]  C. Danilowicz,et al.  Impacts of magnesium ions on the unzipping of λ-phage DNA , 2006 .

[30]  N. Kleckner,et al.  Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division. , 2003, Genes & development.

[31]  R. de Vries,et al.  DNA psi-condensation and reentrant decondensation: effect of the PEG degree of polymerization. , 2005, The journal of physical chemistry. B.

[32]  F. Johnson,et al.  In vivo veritas: using yeast to probe the biological functions of G-quadruplexes. , 2008, Biochimie.

[33]  S. Keeney,et al.  Communication between homologous chromosomes: genetic alterations at a nuclease‐hypersensitive site can alter mitotic chromatin structure at that site both in cis and in trans , 1996, Genes to cells : devoted to molecular & cellular mechanisms.

[34]  N. Kleckner,et al.  Examination of Interchromosomal Interactions in Vegetatively Growing Diploid Schizosaccharomyces pombe Cells by Cre/loxP Site-Specific Recombination , 2008, Genetics.

[35]  A. Kornyshev,et al.  Electrostatic Zipper Motif for DNA Aggregation , 1999 .

[36]  Chia-Lun Tsai,et al.  Transient Homologous Chromosome Pairing Marks the Onset of X Inactivation , 2006, Science.

[37]  R. Dickerson,et al.  DNA structure from A to Z. , 1992, Methods in enzymology.

[38]  R. Podgornik,et al.  A phenomenological one-parameter equation of state for osmotic pressures of PEG and other neutral flexible polymers in good solvents. , 2009, The journal of physical chemistry. B.

[39]  Jeannie T. Lee,et al.  The pluripotency factor, Oct4, interacts with Ctcf and also controls X-chromosome pairing and counting , 2009, Nature.

[40]  Nancy Kleckner,et al.  The structure of DNA overstretched from the 5′5′ ends differs from the structure of DNA overstretched from the 3′3′ ends , 2009, Proceedings of the National Academy of Sciences.

[41]  V A Parsegian,et al.  Direct measurement of the intermolecular forces between counterion-condensed DNA double helices. Evidence for long range attractive hydration forces. , 1992, Biophysical journal.

[42]  Richard Lavery,et al.  Structure and mechanics of single biomolecules: experiment and simulation , 2002 .

[43]  A. Kornyshev,et al.  Structure and interactions of biological helices , 2007 .

[44]  H. Scherthan,et al.  Dynamics of chromosome organization and pairing during meiotic prophase in fission yeast , 1994, The Journal of cell biology.

[45]  M. Prentiss,et al.  Demonstration that the shear force required to separate short double-stranded DNA does not increase significantly with sequence length for sequences longer than 25 base pairs. , 2008, Physical review. E, Statistical, nonlinear, and soft matter physics.

[46]  A. Dernburg,et al.  Prelude to a division. , 2008, Annual review of cell and developmental biology.

[47]  M. Prentiss,et al.  Probing the mechanical stability of DNA in the presence of monovalent cations. , 2008, Journal of the American Chemical Society.

[48]  A. Kornyshev,et al.  Helical coherence of DNA in crystals and solution , 2008, Nucleic acids research.

[49]  Nancy Kleckner,et al.  Chromosome pairing via multiple interstitial interactions before and during meiosis in yeast , 1994, Cell.

[50]  N. Kleckner,et al.  Potential advantages of unstable interactions for pairing of chromosomes in meiotic, somatic, and premeiotic cells. , 1993, Cold Spring Harbor symposia on quantitative biology.

[51]  J. Rossignol,et al.  Gene inactivation triggered by recognition between DNA repeats , 1994, Experientia.

[52]  Peter R Cook,et al.  The role of specialized transcription factories in chromosome pairing. , 2008, Biochimica et biophysica acta.

[53]  S. McGavin,et al.  Models of specifically paired like (homologous) nucleic acid structures. , 1971, Journal of molecular biology.

[54]  N. Hud.  Nucleic acid-metal ion interactions , 2009 .

[55]  V. Parsegian,et al.  Temperature‐induced complementarity as a mechanism for biomolecular assembly , 1994, Proteins.

[56]  V. Bloomfield,et al.  DNA condensation. , 1996, Current opinion in structural biology.

[57]  D. VanDerveer,et al.  Locating monovalent cations in the grooves of B-DNA. , 2001, Biochemistry.

[58]  Jeannie T. Lee,et al.  Evidence that homologous X-chromosome pairing requires transcription and Ctcf protein , 2007, Nature Genetics.

[59]  Willy Wriggers,et al.  Like-charge attraction between polyelectrolytes induced by counterion charge density waves , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[60]  V. Zhurkin,et al.  Modeling DNA deformations. , 2000, Current opinion in structural biology.

[61]  Y. Hiraoka,et al.  A novel meiosis‐specific protein of fission yeast, Meu13p, promotes homologous pairing independently of homologous recombination , 2001, The EMBO journal.