DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

Significance Bacteriophage λ was the first epigenetic switch to be deciphered and continues to contribute to our understanding of gene regulation. Its dormant state is exceptionally stable. In spite of this stability, viral development is efficiently activated in response to DNA damage. This ability to respond efficiently is due to a long-range protein-mediated DNA looping. We developed a single molecule assay based on peptide nucleic acid tethering of a naturally supercoiled DNA plasmid. The internal kinetics of the supercoiled plasmid was monitored, and the dynamics and stability of regulatory protein-mediated DNA looping investigated. We found that the DNA loop becomes tolerant to reductions in the regulator when DNA is supercoiled, thus helping explain the bistable nature of the lambda switch. Bacteriophage λ stably maintains its dormant prophage state but efficiently enters lytic development in response to DNA damage. The mediator of these processes is the λ repressor protein, CI, and its interactions with λ operator DNA. This λ switch is a model on the basis of which epigenetic switch regulation is understood. Using single molecule analysis, we directly examined the stability of the CI-operator structure in its natural, supercoiled state. We marked positions adjacent to the λ operators with peptide nucleic acids and monitored their movement by tethered particle tracking. Compared with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch is significantly increased in the supercoiled system compared with a linear assay, increasing the Hill coefficient.

[1]  Mark Ptashne,et al.  Principles of a switch. , 2011, Nature chemical biology.

[2]  C. Zurla,et al.  The effect of nonspecific binding of lambda repressor on DNA looping dynamics. , 2012, Biophysical journal.

[3]  Laura Finzi,et al.  Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro , 2011, Proceedings of the National Academy of Sciences.

[4]  C. Zurla,et al.  Novel tethered particle motion analysis of CI protein-mediated DNA looping in the regulation of bacteriophage lambda , 2006 .

[5]  T Schlick,et al.  Internal motion of supercoiled DNA: brownian dynamics simulations of site juxtaposition. , 1998, Journal of molecular biology.

[6]  P. Nielsen,et al.  In vitro transcription of a torsionally constrained template. , 2002, Nucleic acids research.

[7]  R. Sternglanz,et al.  Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes , 1982, Cell.

[8]  Kim Sneppen,et al.  Single-cell Analysis of ? Immunity Regulation , 2003 .

[9]  A. D. Kaiser,et al.  Control of lambda repressor synthesis. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[10]  D. Normanno,et al.  Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping , 2008, Nucleic acids research.

[11]  Mark Ptashne,et al.  A Genetic Switch, Phage Lambda Revisited , 2004 .

[12]  A. Ninfa,et al.  DNA supercoiling allows enhancer action over a large distance , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[13]  A. G. Marr,et al.  STUDIES ON THE REPRESSION OF BETA-GALACTOSIDASE IN ESCHERICHIA COLI. , 1964, Biochimica et biophysica acta.

[14]  D. Dunlap,et al.  AFM studies of λ repressor oligomers securing DNA loops , 2009 .

[15]  P. Nielsen,et al.  Enhanced peptide nucleic acid binding to supercoiled DNA: possible implications for DNA "breathing" dynamics. , 1996, Biochemistry.

[16]  J. W. Little,et al.  Stability and Instability in the Lysogenic State of Phage Lambda , 2010, Journal of bacteriology.

[17]  A. G. Marr,et al.  Studies on the repression of β-galactosidase in Escherichia coli , 1964 .

[18]  Franca Esposito,et al.  Supercoiling in prokaryotic and eukaryotic DNA: changes in response to topological perturbation of plasmids in E. coli and SV40 in vitro, in nuclei and in CV-1 cells , 1987, Nucleic Acids Res..

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

[20]  K. Sneppen,et al.  . s of t ] 1 9 O ct 2 00 0 Stability Puzzles in Phage λ , 2008 .

[21]  L. Oddershede,et al.  Stepwise bending of DNA by a single TATA-box binding protein. , 2006, Biophysical Journal.

[22]  Matthew W. Pennington,et al.  Three-dimensional characterization of tethered microspheres by total internal reflection fluorescence microscopy. , 2005, Biophysical journal.

[23]  J. W. Little,et al.  LexA and λ Cl repressors as enzymes: Specific cleavage in an intermolecular reaction , 1993, Cell.

[24]  A. D. Kaiser,et al.  Control of λ Repressor Synthesis , 1971 .

[25]  R. Sinsheimer,et al.  DNA of bacteriophage PM2: a closed circular double-stranded molecule. , 1969, Proceedings of the National Academy of Sciences of the United States of America.

[26]  I. Dodd,et al.  Cooperativity in long-range gene regulation by the lambda CI repressor. , 2004, Genes & development.

[27]  D. Dunlap,et al.  The antiparallel loops in gal DNA , 2008, Nucleic acids research.

[28]  D. Dunlap,et al.  AFM studies of lambda repressor oligomers securing DNA loops. , 2009, Current pharmaceutical biotechnology.

[29]  Benno Müller-Hill,et al.  Four dimers of λ repressor bound to two suitably spaced pairs of λ operators form octamers and DNA loops over large distances , 1999, Current Biology.

[30]  David Bensimon,et al.  Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[31]  A. Vologodskii,et al.  Probability of the site juxtaposition determines the rate of protein-mediated DNA looping. , 2007, Biophysical journal.

[32]  David Dunlap,et al.  Direct demonstration and quantification of long-range DNA looping by the λ bacteriophage repressor , 2009, Nucleic acids research.

[33]  G. K. Ackers,et al.  Quantitative model for gene regulation by lambda phage repressor. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[34]  B. Müller-Hill,et al.  Four dimers of lambda repressor bound to two suitably spaced pairs of lambda operators form octamers and DNA loops over large distances. , 1999, Current biology : CB.

[35]  Haw Yang,et al.  DNA looping can enhance lysogenic CI transcription in phage lambda , 2008, Proceedings of the National Academy of Sciences.

[36]  K. Sneppen,et al.  Single-cell analysis of lambda immunity regulation. , 2003, Journal of molecular biology.

[37]  J. Thompson,et al.  The Role of Coevolution , 2012, Science.

[38]  M. Egholm,et al.  Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. , 1995, Nucleic acids research.

[39]  M. Egholm,et al.  Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. , 1991, Science.

[40]  R. H. Berg,et al.  Solid‐Phase synthesis of peptide nucleic acids , 1995, Journal of peptide science : an official publication of the European Peptide Society.

[41]  Ian B. Dodd,et al.  Cooperativity in long-range gene regulation by the λ CI repressor , 2004 .

[42]  J. W. Little,et al.  LexA and lambda Cl repressors as enzymes: specific cleavage in an intermolecular reaction. , 1993, Cell.