The recombination hotspot Chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5'-GCTGGTGG-3'.

The RecBCD enzyme of Escherichia coli functions in the seemingly disparate roles of homologous recombination and the degradation of DNA. Which of these two roles it assumes is regulated by the 8-base recombination hotspot, Chi. Using double-stranded DNA substrates that are heteroduplex at the Chi locus we have established the determinants for Chi recognition. Our results show that an actively translocating RecBCD enzyme requires only the sequence information in the 5'-GCTGGTGG-3'-containing strand to recognize and to be regulated by Chi. Furthermore, the RecBCD enzyme can translocate through DNA heteroduplex bubbles as large as 22 bases, and still recognize a Chi sequence embedded in this region. This implies that recognition of Chi occurs following the unwinding of the DNA.

[1]  Jeffrey W. Roberts,et al.  Base-Specific Recognition of the Nontemplate Strand of Promoter DNA by E. coli RNA Polymerase , 1996, Cell.

[2]  Jeffrey W. Roberts,et al.  Function of E. coli RNA Polymerase σ Factor- σ70 in Promoter-Proximal Pausing , 1996, Cell.

[3]  S. Kowalczykowski,et al.  Role of the Escherichia coli Recombination Hotspot, χ, in RecABCD-dependent Homologous Pairing (*) , 1995, The Journal of Biological Chemistry.

[4]  P. V. von Hippel,et al.  Coupling of RNA displacement and intrinsic termination in transcription from synthetic RNA.DNA bubble duplex constructs. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  P. Dehaseth,et al.  Mutations in sigma factor that affect the temperature dependence of transcription from a promoter, but not from a mismatch bubble in double-stranded DNA. , 1994, Biochemistry.

[6]  Jeffrey W. Roberts,et al.  Function of a nontranscribed DNA strand site in transcription elongation , 1994, Cell.

[7]  P. Dehaseth,et al.  A mismatch bubble in double-stranded DNA suffices to direct precise transcription initiation by Escherichia coli RNA polymerase. , 1994, The Journal of biological chemistry.

[8]  P. V. von Hippel,et al.  RNA displacement pathways during transcription from synthetic RNA-DNA bubble duplexes. , 1994, Biochemistry.

[9]  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.

[10]  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.

[11]  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.

[12]  P. V. von Hippel,et al.  Functional transcription elongation complexes from synthetic RNA-DNA bubble duplexes. , 1992, Science.

[13]  F. R. Bryant,et al.  Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 2. DNA renaturation reaction. , 1992, Biochemistry.

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

[15]  S. Kowalczykowski,et al.  Homologous pairing in vitro stimulated by the recombination Hotspot, Chi , 1991, Cell.

[16]  S. Kowalczykowski,et al.  RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[17]  S. Kowalczykowski,et al.  Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins. , 1989, The Journal of biological chemistry.

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

[19]  Dickerson.  Nucleic Acids and Molecular Biology , 1987 .

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

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

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

[23]  Gerald R. Smith.  Chi hotspots of generalized recombination , 1983, Cell.

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

[25]  F. Blattner,et al.  Nucleotide sequence of the chi recombinational hot spot chi +D in bacteriophage lambda , 1981, Journal of virology.

[26]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

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

[28]  V. Mackay,et al.  Selective inhibition of the dnase activity of the recBC enzyme by the DNA binding protein from Escherichia coli. , 1976, The Journal of biological chemistry.

[29]  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.

[30]  F. Stahl,et al.  Rec-mediated recombinational hot spot activity in bacteriophage lambda. I. Hot spot activity associated with spi-deletions and bio substitutions. , 1974, Genetics.

[31]  J. Lebowitz,et al.  Physical and Topological Properties of Circular DNA , 1966, The Journal of general physiology.

[32]  M. McPherson.  Directed mutagenesis : a practical approach , 1991 .