Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 ± 172 base pairs per second (0.30 µm s-1), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

[1]  S. Kowalczykowski,et al.  Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot chi in vitro: evidence for functional inactivation or loss of the RecD subunit. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Kowalczykowski,et al.  Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay. , 1989, Biochemistry.

[3]  S. Kowalczykowski,et al.  Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme. , 1992, The Journal of biological chemistry.

[4]  S. Kowalczykowski,et al.  A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands. , 1996, Nucleic acids research.

[5]  G. R. Smith,et al.  Substrate specificity of the DNA unwinding activity of the RecBC enzyme of Escherichia coli. , 1985, Journal of molecular biology.

[6]  Robert Landick,et al.  RNA Polymerase as a Molecular Motor , 1998, Cell.

[7]  A. Kuzminov.  Recombinational Repair of DNA Damage inEscherichia coli and Bacteriophage λ , 1999, Microbiology and Molecular Biology Reviews.

[8]  L. Finzi,et al.  Measurement of lactose repressor-mediated loop formation and breakdown in single DNA molecules , 1995, Science.

[9]  S. Kowalczykowski,et al.  Translocation step size and mechanism of the RecBC DNA helicase , 2000, Nature.

[10]  P. Boehmer,et al.  Escherichia coli RecBCD enzyme: inducible overproduction and reconstitution of the ATP-dependent deoxyribonuclease from purified subunits. , 1991, Gene.

[11]  Christoph F. Schmidt,et al.  Direct observation of kinesin stepping by optical trapping interferometry , 1993, Nature.

[12]  S. Lovett,et al.  The genetic dependence of recombination in recD mutants of Escherichia coli. , 1988, Genetics.

[13]  M. Sheetz,et al.  Transcription by single molecules of RNA polymerase observed by light microscopy , 1991, Nature.

[14]  D E Smith,et al.  Direct observation of tube-like motion of a single polymer chain. , 1994, Science.

[15]  G. R. Smith,et al.  Strand-specific binding to duplex DNA ends by the subunits of the Escherichia coli RecBCD enzyme. , 1993, Journal of molecular biology.

[16]  J. Gelles,et al.  Subunit Interactions in Dimeric Kinesin Heavy Chain Derivatives That Lack the Kinesin Rod , 1995, The Journal of Biological Chemistry.

[17]  S. Kowalczykowski,et al.  The mutant recBCD enzyme, recB2109CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro. , 1993, Journal of molecular biology.

[18]  S. Kowalczykowski,et al.  Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay. , 1998, Nucleic acids research.

[19]  I. Siddiqi,et al.  Further tests of a recombination model in which chi removes the RecD subunit from the RecBCD enzyme of Escherichia coli. , 1990, Genetics.

[20]  R. Sousa,et al.  A model for the mechanism of polymerase translocation. , 1997, Journal of molecular biology.

[21]  S. Kowalczykowski,et al.  The recombination hotspot χ is a regulatory sequence that acts by attenuating the nuclease activity of the E. coli RecBCD enzyme , 1993, Cell.

[22]  R. Landick,et al.  Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule. , 1994, Biophysical journal.

[23]  J. Gelles,et al.  Failure of a single-headed kinesin to track parallel to microtubule protofilaments , 1995, Nature.

[24]  S. Kowalczykowski,et al.  Chi‐activated RecBCD enzyme possesses 5′→3′ nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit , 1997, Genes to cells : devoted to molecular & cellular mechanisms.

[25]  D. Schultz,et al.  RecBC enzyme nicking at chi sites during DNA unwinding: Location and orientation-dependence of the cutting , 1985, Cell.

[26]  David Keller,et al.  Single-molecule studies of the effect of template tension on T7 DNA polymerase activity , 2000, Nature.

[27]  W. Wooster,et al.  Crystal structure of , 2005 .

[28]  I. Lehman,et al.  On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli. , 1977, The Journal of biological chemistry.

[29]  J. Cronan,et al.  Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface. , 1994, The Journal of biological chemistry.

[30]  A. Kuzminov.  Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. , 1999, Microbiology and molecular biology reviews : MMBR.

[31]  S. Kowalczykowski,et al.  A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme* , 1999, The Journal of Biological Chemistry.

[32]  S. Kowalczykowski,et al.  Characterization of the Adenosinetriphosphatase Activity of the Escherichia coli RecBCD Enzyme : Relationship of ATP Hydrolysis to the Unwinding of Duplex DNA + , 1988 .

[33]  J. Cronan.  Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins. , 1990, The Journal of biological chemistry.

[34]  G. R. Smith,et al.  Regulation of homologous recombination: Chi inactivates RecBCD enzyme by disassembly of the three subunits. , 1999, Genes & development.

[35]  R. Balhorn,et al.  Protamine-induced condensation and decondensation of the same DNA molecule. , 1999, Science.

[36]  Edward S. Yeung,et al.  Differences in the chemical reactivity of individual molecules of an enzyme , 1995, Nature.

[37]  W. Wackernagel,et al.  Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[38]  S. Kowalczykowski,et al.  SSB protein controls RecBCD enzyme nuclease activity during unwinding: a new role for looped intermediates. , 1998, Journal of molecular biology.

[39]  B G de Grooth,et al.  Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1. , 1999, Cytometry.

[40]  Gerald R. Smith,et al.  Unwinding and rewinding of DNA by the RecBC enzyme , 1980, Cell.

[41]  S. Kowalczykowski,et al.  Biochemistry of homologous recombination in Escherichia coli. , 1994, Microbiological reviews.