Subject(s)
Disease Models, Animal , Gene Targeting/methods , Genetic Diseases, Inborn/genetics , Genetic Testing/methods , Rats/genetics , Animals , Bacteriophage mu/genetics , Cryopreservation , DNA Transposable Elements/genetics , Ethylnitrosourea/pharmacology , Humans , Male , Mutagenesis, Insertional/methods , Mutation , Sperm Injections, Intracytoplasmic , Spermatozoa/cytologyABSTRACT
Assembly of the Mu transpososome is dependent on specific binding sites for the MuA transposase near the ends of the phage genome. MuA also contacts terminal nucleotides but only upon transpososome assembly, and base-specific recognition of the terminal nucleotides is critical for assembly. We show that Mu ends lacking the terminal 5 bp can form transpososomes, while longer DNA substrates with mutated terminal nucleotides cannot. The impact of the mutations can be suppressed by base mismatches near the end of Mu. Deletion of the flanking strands or mutation of the terminal nucleotides has differential effects on the cleavage and strand transfer reactions. These results show that the terminal nucleotides control the assembly and activation of transpososomes by influencing conformational changes around the active site.
Subject(s)
DNA/genetics , DNA/metabolism , Transposases/genetics , Transposases/metabolism , Bacteriophages/genetics , Base Pair Mismatch , Binding Sites , Fluorescence Resonance Energy Transfer , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Multienzyme Complexes , Mutation , Nucleic Acid Conformation , Nucleotides/genetics , Nucleotides/metabolism , Plasmids/genetics , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombination, Genetic , Transposases/chemistryABSTRACT
Phage Mu DNA transposes to duplex target DNA sites with limited sequence specificity. Here we demonstrate that Mu transposition exhibits a strong target site preference for all single-nucleotide mismatches. This finding has implications for the mechanism of transposition and provides a powerful tool for genomic research. A single mismatch could be detected as a preferred target of Mu transposition in the presence of 300,000-fold excess of nonmismatched sites. We demonstrate the detection of both heterozygous and homozygous mutations in the cystic fibrosis transmembrane conductance regulator gene and single nucleotide polymorphism in HLA region by Mu transposition mismatch analysis procedure.
Subject(s)
Base Pair Mismatch/genetics , Polymorphism, Genetic , Transposases/genetics , Bacteriophages/enzymology , Bacteriophages/genetics , Base Sequence , Chromosome Mapping/methods , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA, Viral/genetics , Humans , Molecular Sequence Data , OligodeoxyribonucleotidesABSTRACT
Clp/Hsp100 ATPases comprise a large family of ATP-dependent chaperones, some of which are regulatory components of two-component proteases. Substrate specificity resides in the Clp protein and the current thinking is that Clp proteins recognize motifs located near one or the other end of the substrate. We tested whether or not ClpA and ClpX can recognize tags when they are located in the interior of the primary sequence of the substrate. A protein with an NH2-terminal ClpA recognition tag, plasmid P1 RepA, was fused to the COOH terminus of green fluorescent protein (GFP). GFP is not recognized by ClpA or ClpX and is not degraded by ClpAP or ClpXP. We found that ClpA binds and unfolds the fusion protein and ClpAP degrades the protein. Both the GFP and RepA portions of the fusion protein are degraded. A protein with a COOH-terminal ClpX tag, MuA, was fused to the NH2 terminus of GFP. ClpXP degrades MuA-GFP, however, the rate is 10-fold slower than that of GFP-MuA. The MuA portion but not the GFP portion of MuA-GFP is degraded. Thus, a substrate with an internal ClpA recognition motif can be unfolded by ClpA and degraded by ClpAP. Similarly, although less efficiently, ClpXP degrades a substrate with an internal ClpX recognition motif. We also found that ClpA recognizes the NH2-terminal 15 aa RepA tag, when it is fused to the COOH terminus of GFP. Moreover, ClpA recognizes the RepA tag in either the authentic or inverse orientation.
