Unwinding of Nucleosomal DNA by a DNA Helicase (*)

We have asked whether a DNA helicase can unwind DNA contained within both isolated native chromatin and reconstituted chromatin containing regularly spaced arrays of nucleosome cores on a linear tandem repeat sequence. We find that Escherichia coli recBCD enzyme is capable of unwinding these DNA substrates and displacing the nucleosomes, although both the rate and the processivity of enzymatic unwinding are inhibited (a maximum of 3- and >25-fold, respectively) as the nucleosome density on the template is increased. The observed rate of unwinding is not affected if the histone octamer is chemically cross-linked; thus, dissociation, or splitting, of the histone octamer is not required for unwinding to occur. The unwinding of native chromatin isolated from HeLa cell nuclei occurs both in the absence and in the presence of linker histone H1. These results suggest that as helicases unwind DNA, they facilitate nuclear processes by acting to clear DNA of histones or DNA-binding proteins in general.

[1]  S. Linn,et al.  Purification and properties of the recBC DNase of Escherichia coli K-12. , 1972, The Journal of biological chemistry.

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

[3]  K. V. van Holde,et al.  DNA and protein determinants of nucleosome positioning on sea urchin 5S rRNA gene sequences in vitro. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Wetmur,et al.  Studies on the noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded nucleic acids. , 1976, Biochemistry.

[5]  A. Wolffe,et al.  Chromatin assembly on replicating DNA in vitro. , 1990, Nucleic acids research.

[6]  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. , 1989, Biochemistry.

[7]  G. Felsenfeld,et al.  Chromatin as an essential part of the transcriptional mechanim , 1992, Nature.

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

[9]  K. V. van Holde,et al.  Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1. , 1989, Biochemistry.

[10]  D. Lohr,et al.  Assembly and structural properties of subsaturated chromatin arrays. , 1993, The Journal of biological chemistry.

[11]  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.

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

[13]  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.

[14]  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.

[15]  E. Bradbury,et al.  Chromatosome positioning on assembled long chromatin. Linker histones affect nucleosome placement on 5 S rDNA. , 1991, Journal of molecular biology.

[16]  MATING-TYPE GENE SWITCHING IN SACCHAROMYCES CEREVISIAE , 1992 .

[17]  E. Bradbury,et al.  Histone octamer dissociation is not required for transcript elongation through arrays of nucleosome cores by phage T7 RNA polymerase in vitro. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Roberge,et al.  Nucleosome arrays inhibit both initiation and elongation of transcripts by bacteriophage T7 RNA polymerase. , 1992, Journal of molecular biology.

[19]  J. Gatewood,et al.  Atomic force microscope measurements of nucleosome cores assembled along defined DNA sequences. , 1993, Biochemistry.

[20]  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.

[21]  J. T. Kadonaga,et al.  Role of nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II. , 1991, Science.

[22]  T. Kelly,et al.  The fate of parental nucleosomes during SV40 DNA replication. , 1992, The Journal of biological chemistry.

[23]  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.

[24]  V. Mackay,et al.  The recBC deoxyribonuclease of Escherichia coli K-12. Substrate specificity and reaction intermediates. , 1973, The Journal of biological chemistry.

[25]  K. Muniyappa,et al.  RecA protein promoted homologous pairing in vitro. Pairing between linear duplex DNA bound to HU Protein (nucleosome cores) and nucleoprotein filaments of recA protein-single-stranded DNA. , 1989, The Journal of biological chemistry.

[26]  S. Kowalczykowski,et al.  Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks chi-specific, but not non-specific, nuclease activity. , 1993, Journal of molecular biology.

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

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

[29]  Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[30]  B. Alberts,et al.  In vitro replication through nucleosomes without histone displacement , 1990, Nature.

[31]  V Jackson,et al.  In vivo studies on the dynamics of histone-DNA interaction: evidence for nucleosome dissolution during replication and transcription and a low level of dissolution independent of both. , 1990, Biochemistry.

[32]  A. Shimamura,et al.  Histone H1 represses transcription from minichromosomes assembled in vitro , 1989, Molecular and cellular biology.

[33]  K. V. van Holde,et al.  What happens to nucleosomes during transcription? , 1992, The Journal of biological chemistry.

[34]  Claudio Nicolini,et al.  Chromatin Structure and Function , 1979, NATO Advanced Study Institutes Series.

[35]  S. Takada,et al.  Alteration of DNA primase activity by phosphorylation and de-phosphorylation of histone H1. , 1989, Biochemical and Biophysical Research Communications - BBRC.

[36]  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.

[37]  C. Georgopoulos,et al.  Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

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

[39]  G. R. Smith,et al.  Distribution of Chi-stimulated recombinational exchanges and heteroduplex endpoints in phage lambda. , 1989, Genetics.

[40]  G. Felsenfeld,et al.  A nucleosome core is transferred out of the path of a transcribing polymerase , 1992, Cell.

[41]  T. Petes,et al.  Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Saccharomyces cerevisiae. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[42]  T. Krude,et al.  Transfer of nucleosomes from parental to replicated chromatin , 1991, Molecular and cellular biology.

[43]  D. Luse,et al.  Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing. , 1991, Genes & development.

[44]  G. R. Smith,et al.  Chromosomal context dependence of a eukaryotic recombinational hot spot. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[45]  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.

[46]  F. Thoma,et al.  Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: A model system for study of higher order structure , 1985, Cell.

[47]  I. Tsaneva,et al.  In vitro transcription through nucleosomes by T7 RNA polymerase. , 1992, EMBO Journal.

[48]  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.

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