Global jumping and domain-specific intersegment transfer between DNA cognate sites of the multidomain transcription factor Oct-1

At high DNA concentration, as found in the nucleus, DNA-binding proteins search for specific binding sites by hopping between separate DNA strands. Here, we use 15Nz-exchange transverse relaxation optimized NMR spectroscopy to characterize the mechanistic details of intermolecular hopping for the multidomain transcription factor, human Oct-1. Oct-1 is a member of the POU family of transcription factors and contains two helix–turn–helix DNA-binding domains, POUHD and POUS, connected by a relatively short flexible linker. The two domains were found to exchange between specific sites at significantly different rates. The cotranscription factor, Sox2, decreases the exchange rate and equilibrium dissociation constant for Oct-1 ≥5-fold and ≈20-fold, respectively, by slowing the exchange rate for the POUS domain. DNA-dependent exchange rates measured at physiological ionic strength indicate that the two domains use both an intersegmental transfer mechanism, which does not involve the intermediary of free protein, and a fully dissociative or jumping mechanism to translocate between cognate sites. These data represent an example of dissecting domain-specific kinetics for protein–DNA association involving a multidomain protein and provide evidence that intersegmental transfer involves a ternary intermediate, or transition state in which the DNA-binding domains bridge two different DNA fragments simultaneously.

[1]  P. V. von Hippel,et al.  Diffusion-driven mechanisms of protein translocation on nucleic acids. 2. The Escherichia coli repressor--operator interaction: equilibrium measurements. , 1981, Biochemistry.

[2]  Antoine M. van Oijen,et al.  A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[3]  P. V. von Hippel,et al.  Diffusion-controlled macromolecular interactions. , 1985, Annual review of biophysics and biophysical chemistry.

[4]  Huan-Xiang Zhou,et al.  Enhancement of association rates by nonspecific binding to DNA and cell membranes. , 2004, Physical review letters.

[5]  H. Mcconnell.  Reaction Rates by Nuclear Magnetic Resonance , 1958 .

[6]  S. Nordeen,et al.  DNA Intersegment Transfer, How Steroid Receptors Search for A Target Site* , 1997, The Journal of Biological Chemistry.

[7]  V. Walsh.  Models and Theory , 1987 .

[8]  A. Riggs,et al.  The lac represser-operator interaction , 1970 .

[9]  S. Halford,et al.  How to get from A to B: strategies for analysing protein motion on DNA , 2002, European Biophysics Journal.

[10]  Mark J Alkema,et al.  The DNA binding specificity of the bipartite POU domain and its subdomains. , 1992, The EMBO journal.

[11]  Stoyan Milev,et al.  Enthalpic and entropic effects of salt and polyol osmolytes on site-specific protein-DNA association: the integrase Tn916-DNA complex. , 2005, Biochemistry.

[12]  P. Privalov,et al.  What drives proteins into the major or minor grooves of DNA? , 2007, Journal of molecular biology.

[13]  G. Clore,et al.  Direct observation of enhanced translocation of a homeodomain between DNA cognate sites by NMR exchange spectroscopy. , 2006, Journal of the American Chemical Society.

[14]  F. Collins,et al.  Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin , 1988, Molecular and cellular biology.

[15]  M. Malamy,et al.  Role of the F factor oriV1 region in recA-independent illegitimate recombination. Stable replicon fusions of the F derivative pOX38 and pBR322-related plasmids. , 1984, Journal of molecular biology.

[16]  Matthias Wilmanns,et al.  Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. , 2003, Genes & development.

[17]  A. Riggs,et al.  The lac repressor-operator interaction. 3. Kinetic studies. , 1970, Journal of molecular biology.

[18]  C. Pabo,et al.  DNA recognition by Cys2His2 zinc finger proteins. , 2000, Annual review of biophysics and biomolecular structure.

[19]  P. V. von Hippel,et al.  Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. , 1981, Biochemistry.

[20]  Fulvio Mavilio,et al.  The Recruitment of SOX/OCT Complexes and the Differential Activity of HOXA1 and HOXB1 Modulate the Hoxb1Auto-regulatory Enhancer Function* , 2001, The Journal of Biological Chemistry.

[21]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[22]  B. Shklovskii,et al.  How a protein searches for its specific site on DNA: the role of intersegment transfer. , 2007, Physical review. E, Statistical, nonlinear, and soft matter physics.

[23]  G. Ulrich Nienhaus,et al.  Protein-Ligand Interactions , 2005, Methods in Molecular Biology™.

[24]  C. Perrin,et al.  Application of two-dimensional NMR to kinetics of chemical exchange , 1990 .

[25]  A M Gronenborn,et al.  Structures of larger proteins in solution: three- and four-dimensional heteronuclear NMR spectroscopy. , 1991, Science.

[26]  G. Clore,et al.  TROSY-based z-exchange spectroscopy: application to the determination of the activation energy for intermolecular protein translocation between specific sites on different DNA molecules. , 2007, Journal of the American Chemical Society.

[27]  J. Elf,et al.  Probing Transcription Factor Dynamics at the Single-Molecule Level in a Living Cell , 2007, Science.

[28]  Huan‐Xiang Zhou,et al.  Disparate ionic‐strength dependencies of on and off rates in protein–protein association , 2001, Biopolymers.

[29]  K Wüthrich,et al.  TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Pabo,et al.  Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage. , 1996, Genes & development.

[31]  Jie Shen,et al.  T to C Substitution at –175 or –173 of the γ-Globin Promoter Affects GATA-1 and Oct-1 Binding in Vitro Differently but Can Independently Reproduce the Hereditary Persistence of Fetal Hemoglobin Phenotype in Transgenic Mice* , 2005, Journal of Biological Chemistry.

[32]  M G Fried,et al.  Kinetics and mechanism in the reaction of gene regulatory proteins with DNA. , 1984, Journal of molecular biology.

[33]  Scot A. Wolfe,et al.  DNA RECOGNITION BY Cys 2 His 2 ZINC FINGER PROTEINS , 2000 .

[34]  E. Reddy,et al.  The myb gene family in cell growth, differentiation and apoptosis , 1999, Oncogene.

[35]  G. Marius Clore,et al.  Molecular Basis for Synergistic Transcriptional Activation by Oct1 and Sox2 Revealed from the Solution Structure of the 42-kDa Oct1·Sox2·Hoxb1-DNA Ternary Transcription Factor Complex* , 2004, Journal of Biological Chemistry.

[36]  G. Marius Clore,et al.  Detecting transient intermediates in macromolecular binding by paramagnetic NMR , 2006, Nature.

[37]  G Marius Clore,et al.  NMR structural and kinetic characterization of a homeodomain diffusing and hopping on nonspecific DNA , 2006, Proceedings of the National Academy of Sciences.

[38]  Juli D. Klemm,et al.  Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules , 1994, Cell.

[39]  M. Rosenfeld,et al.  POU domain factors in the neuroendocrine system: lessons from developmental biology provide insights into human disease. , 2001, Endocrine reviews.

[40]  S. Halford,et al.  Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA. , 2005, Proceedings of the National Academy of Sciences of the United States of America.