Nucleotide shuffling and ssDNA recognition in Oxytricha nova telomere end‐binding protein complexes

Sequence‐specific protein recognition of single‐stranded nucleic acids is critical for many fundamental cellular processes, such as DNA replication, DNA repair, transcription, translation, recombination, apoptosis and telomere maintenance. To explore the mechanisms of sequence‐specific ssDNA recognition, we determined the crystal structures of 10 different non‐cognate ssDNAs complexed with the Oxytricha nova telomere end‐binding protein (OnTEBP) and evaluated their corresponding binding affinities (PDB ID codes 1PH1–1PH9 and 1PHJ). The thermodynamic and structural effects of these sequence perturbations could not have been predicted based solely upon the cognate structure. OnTEBP accommodates non‐cognate nucleotides by both subtle adjustments and surprisingly large structural rearrangements in the ssDNA. In two complexes containing ssDNA intermediates that occur during telomere extension by telomerase, entire nucleotides are expelled from the complex. Concurrently, the sequence register of the ssDNA shifts to re‐establish a more cognate‐like pattern. This phenomenon, termed nucleotide shuffling, may be of general importance in protein recognition of single‐stranded nucleic acids. This set of structural and thermodynamic data highlights a fundamental difference between protein recognition of ssDNA versus dsDNA.

[1]  S. Stevens,et al.  Evidence for sequence-specific recognition of DNA by anti-single-stranded DNA autoantibodies. , 1999, Biochemistry.

[2]  T. Cech,et al.  Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit. , 1993, Genes & development.

[3]  A. Krainer,et al.  Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA. , 1999, Genes & development.

[4]  R. Darnell,et al.  Sequence-Specific RNA Binding by a Nova KH Domain Implications for Paraneoplastic Disease and the Fragile X Syndrome , 2000, Cell.

[5]  L. Beigelman,et al.  Deletion of a single hydrogen bonding atom from the MS2 RNA operator leads to dramatic rearrangements at the RNA-coat protein interface. , 2000, Nucleic acids research.

[6]  M C Mossing,et al.  Thermodynamic origins of specificity in the lac repressor-operator interaction. Adaptability in the recognition of mutant operator sites. , 1985, Journal of molecular biology.

[7]  C. Harley,et al.  Extension of life-span by introduction of telomerase into normal human cells. , 1998, Science.

[8]  Kazuki Kurimoto,et al.  Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein , 1999, Nature.

[9]  Hidekazu Tanaka,et al.  Involvement of a single‐stranded DNA binding protein, ssCRE‐BP/Purα, in morphine dependence , 1996 .

[10]  Rahul C. Deo,et al.  Recognition of Polyadenylate RNA by the Poly(A)-Binding Protein , 1999, Cell.

[11]  R. S. Spolar,et al.  Coupling of local folding to site-specific binding of proteins to DNA. , 1994, Science.

[12]  R M Esnouf,et al.  Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. , 1999, Acta crystallographica. Section D, Biological crystallography.

[13]  Deborah S Wuttke,et al.  Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13. , 2003, Biochemistry.

[14]  D. Prescott,et al.  All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[15]  C. Prives,et al.  Complex formation between p53 and replication protein A inhibits the sequence-specific DNA binding of p53 and is regulated by single-stranded DNA , 1997, Molecular and cellular biology.

[16]  T. Cech,et al.  Oxytricha telomeric nucleoprotein complexes reconstituted with synthetic DNA. , 1989, Nucleic acids research.

[17]  I. Pérez,et al.  Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo. , 1997, RNA.

[18]  M. Kurpiewski,et al.  The energetic basis of specificity in the Eco RI endonuclease--DNA interaction. , 1990, Science.

[19]  L. E. Engler,et al.  Specific binding by EcoRV endonuclease to its DNA recognition site GATATC. , 1997, Journal of molecular biology.

[20]  A. Zahler,et al.  Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linear DNA molecules. , 1988, Nucleic acids research.

[21]  T R Hughes,et al.  Cdc13p: A Single-Strand Telomeric DNA-Binding Protein with a Dual Role in Yeast Telomere Maintenance , 1996, Science.

[22]  Stephen L. Gasior,et al.  Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[23]  M. Horvath,et al.  Crystal Structure of the Oxytricha nova Telomere End Binding Protein Complexed with Single Strand DNA , 1998, Cell.

[24]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[25]  Deborah S Wuttke,et al.  Homology among telomeric end-protection proteins. , 2003, Structure.

[26]  T. Cech,et al.  Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha. , 1998, Genes & development.

[27]  J M Berger,et al.  The structural basis for terminator recognition by the Rho transcription termination factor. , 1999, Molecular cell.

[28]  T. Cech,et al.  Telomeric DNA-protein interactions of Oxytricha macronuclear DNA. , 1987, Genes & development.

[29]  V. Lundblad,et al.  Cdc13 Delivers Separate Complexes to the Telomere for End Protection and Replication , 2001, Cell.

[30]  D. Gottschling,et al.  Telomere proteins: Specific recognition and protection of the natural termini of Oxytricha macronuclear DNA , 1986, Cell.

[31]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[32]  T. Cech,et al.  Effect of monovalent cation-induced telomeric DNA structure on the binding of Oxytricha telomeric protein. , 1990, Nucleic acids research.

[33]  G. Gallia,et al.  Purα: A multifunctional single-stranded DNA- and RNA-binding protein , 2000 .

[34]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[35]  H. Hohjoh,et al.  Sequence‐specific single‐strand RNA binding protein encoded by the human LINE‐1 retrotransposon , 1997, The EMBO journal.

[36]  D. Rau,et al.  Water release associated with specific binding of gal repressor. , 1995, The EMBO journal.

[37]  K Furukawa,et al.  Thermodynamics of specific and non-specific DNA binding by the c-Myb DNA-binding domain. , 1998, Journal of molecular biology.

[38]  Deborah S Wuttke,et al.  Nucleic acid recognition by OB-fold proteins. , 2003, Annual review of biophysics and biomolecular structure.

[39]  P. Baumann,et al.  Pot1, the Putative Telomere End-Binding Protein in Fission Yeast and Humans , 2001, Science.

[40]  P. V. Hippel,et al.  Protein-DNA recognition: new perspectives and underlying themes , 1994 .

[41]  B. Stillman,et al.  The DNA replication fork in eukaryotic cells. , 1998, Annual review of biochemistry.

[42]  M. Green,et al.  Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal. , 2000, RNA.

[43]  T. Cech,et al.  Characterization of a G-quartet formation reaction promoted by the beta-subunit of the Oxytricha telomere-binding protein. , 1993, Biochemistry.

[44]  A. Antson,et al.  Specificity of TRAP-RNA interactions: crystal structures of two complexes with different RNA sequences. , 2002, Acta crystallographica. Section D, Biological crystallography.

[45]  C B Harley,et al.  Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. , 1993, American journal of human genetics.

[46]  T. Hughes,et al.  Conserved Structure for Single-Stranded Telomeric DNA Recognition , 2002, Science.

[47]  S. Melcher,et al.  Thermodynamics of the interactions of lac repressor with variants of the symmetric lac operator: effects of converting a consensus site to a non-specific site. , 1997, Journal of molecular biology.

[48]  M. Negroni,et al.  Mechanisms of retroviral recombination. , 2001, Annual review of genetics.

[49]  M. Rosbash,et al.  The Splicing Factor BBP Interacts Specifically with the Pre-mRNA Branchpoint Sequence UACUAAC , 1997, Cell.

[50]  D. Rau,et al.  Differences in water release for the binding of EcoRI to specific and nonspecific DNA sequences. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[51]  T. Cech,et al.  Sequence-specific and 3'-end selective single-strand DNA binding by the Oxytricha nova telomere end binding protein alpha subunit. , 2003, Biochemistry.

[52]  G. Clore,et al.  Molecular basis of sequence‐specific single‐stranded DNA recognition by KH domains: solution structure of a complex between hnRNP K KH3 and single‐stranded DNA , 2002, The EMBO journal.

[53]  A. Mondragón,et al.  Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule , 2001, Nature.

[54]  O. Peersen,et al.  Dimeric structure of the Oxytricha nova telomere end-binding protein α-subunit bound to ssDNA , 2002, Nature Structural Biology.

[55]  S. Classen,et al.  Crystal structure of the N-terminal domain of Oxytricha nova telomere end-binding protein alpha subunit both uncomplexed and complexed with telomeric ssDNA. , 2001, Journal of molecular biology.

[56]  Fernando de la Cruz,et al.  The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase , 2001, Nature.

[57]  P. Ross,et al.  Thermodynamics of Cro protein-DNA interactions. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[58]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[59]  S C Schultz,et al.  DNA G-quartets in a 1.86 A resolution structure of an Oxytricha nova telomeric protein-DNA complex. , 2001, Journal of molecular biology.

[60]  R. B. Greaves,et al.  Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA , 1999, Nature.

[61]  T. Cech,et al.  The β subunit of Oxytricha telomere-binding protein promotes G-quartet formation by telomeric DNA , 1993, Cell.

[62]  M. Yamaizumi,et al.  Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[63]  Ana C. Messias,et al.  Structural Basis for Recognition of the Intron Branch Site RNA by Splicing Factor 1 , 2001, Science.

[64]  Wen-Hwa Lee,et al.  BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure. , 2002, Science.

[65]  M. Record,et al.  Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA. , 1991, Methods in enzymology.