ABSTRACT
The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.
Subject(s)
Endodeoxyribonucleases/chemistry , Protein Conformation , Protein Structure, Secondary , Amino Acid Sequence , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Humans , Models, Molecular , Mutation , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins , Spectrum Analysis , Structure-Activity Relationship , X-Ray DiffractionABSTRACT
The signal recognition particle (SRP), a protein-RNA complex conserved in all three kingdoms of life, recognizes and transports specific proteins to cellular membranes for insertion or secretion. We describe here the 1.8 angstrom crystal structure of the universal core of the SRP, revealing protein recognition of a distorted RNA minor groove. Nucleotide analog interference mapping demonstrates the biological importance of observed interactions, and genetic results show that this core is functional in vivo. The structure explains why the conserved residues in the protein and RNA are required for SRP assembly and defines a signal sequence recognition surface composed of both protein and RNA.
Subject(s)
Bacterial Proteins/chemistry , Escherichia coli Proteins , RNA, Bacterial/chemistry , Signal Recognition Particle/chemistry , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Pairing , Binding Sites , Cell Membrane/metabolism , Crystallography, X-Ray , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Guanosine Triphosphate/metabolism , Hydrogen Bonding , Magnesium/metabolism , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Potassium/metabolism , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Signal Recognition Particle/metabolism , Transformation, Bacterial , Water/metabolismABSTRACT
Metal ions are essential for the folding and activity of large catalytic RNAs. While divalent metal ions have been directly implicated in RNA tertiary structure formation, the role of monovalent ions has been largely unexplored. Here we report the first specific monovalent metal ion binding site within a catalytic RNA. As seen crystallographically, a potassium ion is coordinated immediately below AA platforms of the Tetrahymena ribozyme P4-P6 domain, including that within the tetraloop receptor. Interference and kinetic experiments demonstrate that potassium ion binding within the tetraloop receptor stabilizes the folding of the P4-P6 domain and enhances the activity of the Azoarcus group I intron. Since a monovalent ion binding site is integral to the tetraloop receptor, a tertiary structural motif that occurs frequently in RNA, monovalent metal ions are likely to participate in the folding and activity of a wide diversity of RNAs.
Subject(s)
Adenine/chemistry , Cations, Monovalent/chemistry , Nucleic Acid Conformation , RNA, Catalytic/chemistry , Animals , Binding Sites , Cesium/chemistry , Crystallography, X-Ray , Guanosine/analogs & derivatives , Guanosine/chemistry , Potassium/chemistry , RNA Splicing , RNA, Protozoan/chemistry , Tetrahymena , Thallium/chemistry , Thionucleosides/chemistryABSTRACT
Amino acid replacements were made at the interface between two autonomous folding units in the alpha subunit of tryptophan synthase from Salmonella typhimurium to test their mutual interaction energy. The results of equilibrium studies of the urea-induced unfolding reaction of the wild-type and mutant proteins in which phenylalanine 22 is replaced by leucine, isoleucine, and valine can be understood in terms of a selective decrease in the interaction energy between the two folding units; the intrinsic stability of each folding unit is not significantly altered. Kinetic studies of the rate-limiting step in unfolding show that the interaction energy appears in the transition state preceding the native conformation. Comparisons of the individual effects of these nonpolar side chains show that both hydrophobic and steric effects play important roles in the interaction energy between the folding units. The implication of these results is that the high cooperativity observed in the folding of many globular proteins may be reduced by appropriate amino acid replacements.
