How RecBCD Enzyme and Chi Promote DNA Break Repair and Recombination: a Molecular Biologist's View

SUMMARY The repair of DNA double-strand breaks (DSBs) is essential for cell viability and important for homologous genetic recombination. In enteric bacteria such as Escherichia coli, the major pathway of DSB repair requires the RecBCD enzyme, a complex helicase-nuclease regulated by a simple unique DNA sequence called Chi. How Chi regulates RecBCD has been extensively studied by both genetics and biochemistry, and two contrasting mechanisms to generate a recombinogenic single-stranded DNA tail have been proposed: the nicking of one DNA strand at Chi versus the switching of degradation from one strand to the other at Chi. Which of these reactions occurs in cells has remained unproven because of the inability to detect intracellular DNA intermediates in bacterial recombination and DNA break repair. Here, I discuss evidence from a combination of genetics and biochemistry indicating that nicking at Chi is the intracellular (in vivo) reaction. This example illustrates the need for both types of analysis (i.e., molecular biology) to uncover the mechanism and control of complex processes in living cells.

[1]  E. Signer,et al.  The Role of Recombination in Growth of Bacteriophage Lambda II. Inhibition of Growth by Prophage P2 , 1971 .

[2]  V. Simmon,et al.  Degradation of Bacteriophage Lambda Deoxyribonucleic Acid After Restriction by Escherichia coli K-12 , 1972, Journal of bacteriology.

[3]  Piero R Bianco,et al.  Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after chi recognition. , 2005, Molecular cell.

[4]  Rod Balhorn,et al.  Processive translocation and DNA unwinding by individual RecBCD enzyme molecules , 2001, Nature.

[5]  G. R. Smith,et al.  Recombinational hotspot activity of Chi-like sequences. , 1984, Journal of molecular biology.

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

[7]  Ronald J. Baskin,et al.  A Molecular Throttle The Recombination Hotspot χ Controls DNA Translocation by the RecBCD Helicase , 2003, Cell.

[8]  F. Stahl,et al.  Orientation-dependent recombination hotspot activity in bacteriophage λ☆ , 1979 .

[9]  Y. Sakaki,et al.  Purification and properties of the gamma-protein specified by bacteriophage lambda: an inhibitor of the host RecBC recombination enzyme. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[10]  T. Colbert,et al.  Genomics, Chi sites and codons: 'islands of preferred DNA pairing' are oceans of ORFs. , 1998, Trends in genetics : TIG.

[11]  T. Shibata,et al.  Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[12]  K. Brooks,et al.  Behavior of λ Bacteriophage in a Recombination Deficient Strain of Escherichia coli , 1967 .

[13]  G. R. Smith,et al.  Action of RecBCD enzyme on cruciform DNA. , 1990, Journal of molecular biology.

[14]  F. Stahl,et al.  Directionality and nonreciprocality of Chi-stimulated recombination in phage lambda. , 1980, Genetics.

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

[16]  S. Barbour.  Biochemical and genetic studies of recombination proficiency in Escherichia coli K12 , 1972, Molecular and General Genetics MGG.

[17]  W. Franklin,et al.  On the Clustered Exchanges of the RecBCD Pathway Operating on Phage h , 2022 .

[18]  S. Ehrlich,et al.  Chi sequence protects against RecBCD degradation of DNA in vivo. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[19]  S. Kowalczykowski,et al.  The RecA binding locus of RecBCD is a general domain for recruitment of DNA strand exchange proteins. , 2006, Molecular cell.

[20]  B. Alberts.  The Cell as a Collection of Protein Machines: Preparing the Next Generation of Molecular Biologists , 1998, Cell.

[21]  M. Oishi,et al.  An ATP-dependent deoxyribonuclease from Escherichia coli with a possible role in genetic recombination. , 1969, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A J CLARK,et al.  ISOLATION AND CHARACTERIZATION OF RECOMBINATION-DEFICIENT MUTANTS OF ESCHERICHIA COLI K12. , 1965, Proceedings of the National Academy of Sciences of the United States of America.

[23]  G. R. Smith,et al.  recD: the gene for an essential third subunit of exonuclease V. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Daniel G. Anderson,et al.  Reconstitution of an SOS Response Pathway Derepression of Transcription in Response to DNA Breaks , 1998, Cell.

[25]  A. Gruss,et al.  Codon usage can explain GT‐rich islands surrounding Chi sites on the Escherichia coli genome , 1998, Molecular microbiology.

[26]  J. Gelles,et al.  χ-Sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules , 2001, Nature.

[27]  Daniel G. Anderson,et al.  The Translocating RecBCD Enzyme Stimulates Recombination by Directing RecA Protein onto ssDNA in a χ-Regulated Manner , 1997, Cell.

[28]  Gerald R. Smith,et al.  Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots. , 2007, Genes & development.

[29]  F. Korangy,et al.  Kinetics and processivity of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli. , 1993, Biochemistry.

[30]  A. Clark.  Recombination deficient mutants of E. coli and other bacteria. , 1973, Annual review of genetics.

[31]  K. V. SRINATH,et al.  Mechanism of Crossing-over , 1946, Nature.

[32]  A. Clark,et al.  Biochemical and genetic studies of recombination proficiency in Escherichia coli. I. Enzymatic activity associated with recB+ and recC+ genes. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

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

[34]  J. Hurwitz,et al.  The isolation and characterization from Escherichia coli of an adenosine triphosphate-dependent deoxyribonuclease directed by rec B, C genes. , 1971, The Journal of biological chemistry.

[35]  F. Stahl,et al.  Chi and the RecBC D enzyme of Escherichia coli. , 1994, Annual review of genetics.

[36]  N. Willetts,et al.  Characteristics of Some Multiply Recombination-Deficient Strains of Escherichia coli , 1969, Journal of bacteriology.

[37]  Frank Dieterich,et al.  Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica. , 2004, FEMS microbiology letters.

[38]  Lei Zhang.  Molecular Biology of the Cell (2nd edn.) : B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts and J.D. Watson (Eds.), Garland Publishing, Inc., New York, 1989, ISBN 0-8240-3694-4, xxxviii + 1291 pp., US$99.95. , 1994 .

[39]  M. Yu,et al.  Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme. , 1998, Journal of molecular biology.

[40]  P Howard-Flanders,et al.  Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. , 1966, Genetics.

[41]  M. Visnapuu,et al.  Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase , 2010, Nature.

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

[43]  D. Nathans,et al.  Differential synthesis of bacteriophage-specific proteins in MS2-infected Escherichia coli treated with actinomycin. , 1966, Journal of molecular biology.

[44]  S. Kowalczykowski,et al.  The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme. , 1997, Genes & development.

[45]  G. R. Smith,et al.  Escherichia coli RecBC pseudorevertants lacking chi recombinational hotspot activity , 1983, Journal of bacteriology.

[46]  Grace Jordison.  Molecular Biology of the Gene , 1965, The Yale Journal of Biology and Medicine.

[47]  G. R. Smith,et al.  Activity of Chi recombinational hotspots in Salmonella typhimurium. , 1986, Genetics.

[48]  V. Mackay,et al.  The mechanism of degradation of duplex deoxyribonucleic acid by the recBC enzyme of Escherichia coli K-12. , 1974, The Journal of biological chemistry.

[49]  F. Stahl,et al.  Does Chi give or take? , 1984, Genetics.

[50]  D. Thaler,et al.  Double-chain-cut sites are recombination hotspots in the Red pathway of phage lambda. , 1987, Journal of molecular biology.

[51]  I. Kobayashi,et al.  Type III Restriction Is Alleviated by Bacteriophage (RecE) Homologous Recombination Function but Enhanced by Bacterial (RecBCD) Function , 2005, Journal of bacteriology.

[52]  M. Yu,et al.  The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from Escherichia coli. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[53]  D. Catcheside,et al.  Regulation of recombination at the his-3 locus in Neurospora crassa. , 1970, Australian journal of biological sciences.

[54]  T. Horii,et al.  Characteristics of purified recA protein and the regulation of its synthesis in vivo. , 1979, Cold Spring Harbor symposia on quantitative biology.

[55]  B. Michel,et al.  Recombination proteins and rescue of arrested replication forks. , 2007, DNA repair.

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

[57]  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.

[58]  F. Chédin,et al.  The Recombination Hot Spot Chi Is Embedded within Islands of Preferred DNA Pairing Sequences in the E. coli Genome , 1997, Cell.

[59]  J. Lederberg,et al.  Gene Recombination in Escherichia Coli , 1946, Nature.

[60]  S. Hill,et al.  On the clustered exchanges of the RecBCD pathway operating on phage lambda. , 1995, Genetics.

[61]  Steven M Block,et al.  Forward and reverse motion of single RecBCD molecules on DNA. , 2004, Biophysical journal.

[62]  Gerald R. Smith,et al.  Chi Hotspot Activity in Escherichia coli Without RecBCD Exonuclease Activity: Implications for the Mechanism of Recombination , 2007, Genetics.

[63]  J E Wilson,et al.  Chelation of divalent cations by ATP, studied by titration calorimetry. , 1991, Analytical biochemistry.

[64]  E. Signer,et al.  Recombination in bacteriophage lambda. I. Mutants deficient in general recombination. , 1968, Journal of molecular biology.

[65]  E. Signer,et al.  Recombination in bacteriophage λ: I. Mutants deficient in general recombination☆ , 1968 .

[66]  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.

[67]  Gerald R. Smith,et al.  Strand Specificity of Nicking of DNA at Chi Sites by RecBCD Enzyme , 1995, The Journal of Biological Chemistry.

[68]  G. R. Smith,et al.  Genetic functions promoting homologous recombination in Escherichia coli: a study of inversions in phage lambda. , 1987, Genetics.

[69]  G. R. Smith,et al.  A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[70]  D. Chattoraj,et al.  Hotspots for generalized recombination in the Escherichia coli chromosome. , 1978, Journal of molecular biology.

[71]  S. Kowalczykowski,et al.  The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3'. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[72]  G. R. Smith,et al.  Conservation of Chi cutting activity in terrestrial and marine enteric bacteria. , 1986, Journal of molecular biology.

[73]  Gerald R. Smith,et al.  Chi sites, RecBC enzyme, and geneneralized recombination , 1981 .

[74]  N. Cook,et al.  DNA binding to RecD: role of the 1B domain in SF1B helicase activity , 2008, The EMBO journal.

[75]  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.

[76]  D. Chattoraj,et al.  Identity of a Chi site of Escherichia coli and Chi recombinational hotspots of bacteriophage lambda. , 1982, Journal of molecular biology.

[77]  Edward J. Wood,et al.  Biochemistry (3rd ed.) , 2004 .

[78]  E. Goldberg,et al.  Protection of parental T4 DNA from a restriction exonuclease by the product of gene 2. , 1977, Journal of molecular biology.

[79]  A. Goldman,et al.  Meiotic recombination hotspots. , 1995, Annual review of genetics.

[80]  Y. Sakaki,et al.  Purification and Properties of the γ-Protein Specified by Bacteriophage λ: An Inhibitor of the Host RecBC Recombination Enzyme , 1973 .

[81]  O. Bénichou,et al.  Kinetics of target site localization of a protein on DNA: a stochastic approach. , 2004, Biophysical journal.

[82]  D. Henderson,et al.  Recombination-deficient deletions in bacteriophage lambda and their interaction with chi mutations. , 1975, Genetics.

[83]  S. Linn,et al.  A unified mechanism for the nuclease and unwinding activities of the recBC enzyme of Escherichia coli. , 1982, The Journal of biological chemistry.

[84]  I. Kobayashi,et al.  Contribution of RecFOR machinery of homologous recombination to cell survival after loss of a restriction-modification gene complex. , 2009, Microbiology.

[85]  M. Meselson,et al.  Single burst study of rec- and red-mediated recombination in bacteriophage lambda. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

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

[87]  M. Radman,et al.  The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants , 1989, Nature.

[88]  F. Stahl.  Special sites in generalized recombination. , 1979, Annual review of genetics.

[89]  C. Radding.  Regulation of lambda exonuclease. I. Properties of lambda exonuclease purified from lysogens of lambda T11 and wild type. , 1966, Journal of molecular biology.

[90]  F. Stahl,et al.  Rec-mediated recombinational hot spot activity in bacteriophage lambda. III. Chi mutations are site-mutations stimulating rec-mediated recombination. , 1975, Journal of molecular biology.

[91]  A. Clark,et al.  The RecE Pathway of Bacterial Recombination , 1974 .

[92]  G. Weinstock,et al.  Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[93]  K. Mather.  Genetic Recombination , 1960, Nature.

[94]  D. Schultz,et al.  Chi-dependent DNA strand cleavage by RecBC enzyme , 1985, Cell.

[95]  T. Alatossava,et al.  Manipulation of intracellular magnesium content in polymyxin B nonapeptide-sensitized Escherichia coli by ionophore A23187 , 1985, Journal of bacteriology.

[96]  Ilya J. Finkelstein,et al.  Single molecule studies of homologous recombination. , 2008, Molecular bioSystems.

[97]  S. Schbath,et al.  DNA motifs that sculpt the bacterial chromosome , 2010, Nature Reviews Microbiology.

[98]  N. W. Davis,et al.  The complete genome sequence of Escherichia coli K-12. , 1997, Science.

[99]  J. Davies,et al.  Molecular Biology of the Cell , 1983, Bristol Medico-Chirurgical Journal.

[100]  J. Rabinowitz,et al.  Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia coli , 2009, Nature chemical biology.

[101]  E. Signer,et al.  The Role of Recombination in Growth of Bacteriophage Lambda I. The Gamma Gene , 1971 .

[102]  Behavior of lambda bacteriophage in a recombination deficienct strain of Escherichia coli. , 1967, Journal of virology.

[103]  Dale B. Wigley,et al.  Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks , 2004, Nature.

[104]  Gerald R. Smith,et al.  A domain of RecC required for assembly of the regulatory RecD subunit into the Escherichia coli RecBCD holoenzyme. , 2002, Genetics.

[105]  G. R. Smith,et al.  RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[106]  I. Kobayashi,et al.  CHI-STIMULATED RECOMBINATION BETWEEN PHAGE Λ AND THE PLASMID Λdv , 1982 .

[107]  Jody Hey,et al.  What's So Hot about Recombination Hotspots? , 2004, PLoS biology.

[108]  F. Stahl,et al.  Recombination pathway specificity of Chi. , 1977, Genetics.

[109]  B. Ames,et al.  Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. , 1982, The Journal of biological chemistry.

[110]  S. Kowalczykowski,et al.  RecBCD Enzyme and the Repair of Double-Stranded DNA Breaks , 2008, Microbiology and Molecular Biology Reviews.

[111]  R. G. Lloyd,et al.  Targeting Holliday Junctions by the RecG Branch Migration Protein of Escherichia coli * , 1998, The Journal of Biological Chemistry.

[112]  M. Delbrück,et al.  Induced mutations in bacterial viruses. , 1946 .

[113]  G. R. Smith,et al.  Activation of Chi recombinational hotspots by RecBCD-like enzymes from enteric bacteria. , 1989, Journal of molecular biology.

[114]  S. Kunes,et al.  Structure of chi hotspots of generalized recombination , 1981, Cell.

[115]  L. Enquist,et al.  Replication of bacteriophage lambda DNA dependent on the function of host and viral genes. I. Interaction of red, gam and rec. , 1973, Journal of molecular biology.

[116]  I. Kobayashi,et al.  A novel, 11 nucleotide variant of χ, χ∗: one of a class of sequences defining the Escherichia coli recombination hotspot χ , 2000 .

[117]  F. Stahl,et al.  Rec-mediated recombinational hot spot activity in bacteriophage λ , 1974, Molecular and General Genetics MGG.

[118]  H. Echols,et al.  Mutants of bacteriophage λ defective in vegetative genetic recombination , 1968 .

[119]  A. Kuzminov,et al.  Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease. , 1994, The EMBO journal.

[120]  H. Gutz.  Site Specific Induction of Gene Conversion in SCHIZOSACCHAROMYCES POMBE. , 1971, Genetics.

[121]  I. Kobayashi,et al.  Orientation of cohesive end site cos determines the active orientation of chi sequence in stimulating recA . recBC-mediated recombination in phage lambda lytic infections. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[122]  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.

[123]  S. Kowalczykowski,et al.  The RecBC enzyme loads RecA protein onto ssDNA asymmetrically and independently of chi, resulting in constitutive recombination activation. , 1999, Genes & development.

[124]  J. Cleaver,et al.  Solubility and dialysis limits of DNA oligonucleotides. , 1972, Biochimica et biophysica acta.

[125]  S. Ehrlich,et al.  The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro. , 2000, Journal of molecular biology.

[126]  M. Chamberlin,et al.  Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli K12. , 1966, Journal of molecular biology.

[127]  G. Buttin,et al.  Enzymatic DNA degradation in E. coli: its relationship to synthetic processes at the chromosome level. , 1968, Cold Spring Harbor symposia on quantitative biology.

[128]  E. Kellenberger,et al.  High magnesium content ofEscherichia coli B , 2005, Experientia.

[129]  C. Radding.  Regulation of λ exonuclease: I. Properties of λ exonuclease purified from lysogens of λT11 and wild type , 1966 .

[130]  I. Kobayashi,et al.  Coupling with packaging explains apparent nonreciprocality of Chi-stimulated recombination of bacteriophage lambda by RecA and RecBC functions. , 1984, Genetics.

[131]  A. D. Hershey.  SPONTANEOUS MUTATIONS IN BACTERIAL VIRUSES , 1946 .