ABSTRACT
In the presence of ATP and Mg(2+), the bacterial transposon Tn7 translocates via a cut and paste mechanism executed by the transposon-encoded proteins TnsA+TnsB+TnsC+TnsD. We report here that in the presence of Mn(2+), TnsA+TnsB alone can execute the DNA breakage and joining reactions of Tn7 recombination. ATP is not essential in this minimal system, revealing that this cofactor is not directly involved in the chemical steps of recombination. In both the TnsAB and TnsABC+D systems, recombination initiates with double-strand breaks at each transposon end that cut Tn7 away from flanking donor DNA. In the minimal system, breakage occurs predominantly at a single transposon end and the subsequent end-joining reactions are intramolecular, with the exposed 3' termini of a broken transposon end joining near the other end of the Tn7 element in the same donor molecule to form circular transposon species. In contrast, in TnsABC+D recombination, breaks occur at both ends of Tn7 and the two ends join to a target site on a different DNA molecule to form an intermolecular simple insertion. This demonstration of the capacity of TnsAB to execute breakage and joining reactions supports the view that these proteins form the Tn7 transposase.
Subject(s)
Bacterial Proteins/metabolism , DNA Transposable Elements/genetics , DNA, Circular/genetics , DNA-Binding Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/genetics , Recombination, Genetic/genetics , Base Sequence , Cations, Divalent/pharmacology , DNA Probes , DNA Transposable Elements/physiology , DNA, Circular/isolation & purification , DNA, Circular/metabolism , DNA, Circular/ultrastructure , DNA, Superhelical/genetics , DNA, Superhelical/isolation & purification , DNA, Superhelical/metabolism , DNA, Superhelical/ultrastructure , Escherichia coli/enzymology , Manganese/pharmacology , Microscopy, Electron , Molecular Weight , Mutation/drug effects , Mutation/genetics , Nucleic Acid Conformation , Nucleotides/genetics , Recombination, Genetic/drug effectsABSTRACT
A robust Tn7-based in vitro transposition system is described that displays little target site selectivity, allowing the efficient recovery of many different transposon insertions in target DNAs ranging from small plasmids to cosmids to whole genomes. Two miniTn7 derivatives are described that are useful for the analysis of genes: one a derivative for making translational and transcriptional target gene fusions and the other a derivative that can generate 15 bp (5 amino acid) insertions in target DNAs (proteins).
Subject(s)
DNA Transposable Elements , Genes, Bacterial , Genome, Bacterial , Amino Acid Sequence , Base Sequence , Gene Targeting , Molecular Sequence DataABSTRACT
The bacterial transposon Tn7 uses a cut and paste mechanism to translocate between non-homologous insertion sites. In the first step of recombination, double-strand breaks at each transposon end disconnect the element from the donor backbone; in the second step, the now exposed 3' transposon ends join to the target DNA. To dissect the chemical steps in these reactions, we have used mutant transposons altered at and near their extreme termini. We find that the initiating double-strand breaks result from a collaboration of two distinct DNA strand processing activities, one mediating cleavages at the 3' ends of Tn7, which can be blocked by changes at the transposon tips, and another mediating cleavages at the 5' ends. The joining of exposed 3'transposon ends to the target DNA can be blocked by changing the transposon tips. Our results suggest that the target joining step occurs through two usually concerted, but actually separable, reactions in which individual 3' transposon ends are joined to separate strands of the target DNA. Thus Tn7 transposition involves several distinct DNA processing reactions: strand cleavage and strand transfer reactions at the 3' ends of the transposon, and separate strand cleavage reactions at the 5' ends of the transposon.
Subject(s)
DNA Transposable Elements/physiology , DNA/metabolism , Base Sequence , Binding Sites , Models, Genetic , Molecular Sequence Data , Recombination, Genetic/physiologyABSTRACT
Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication. This suggests that RecA protein may be required for correct timing of initiation of DNA replication; however, we show here that initiation of DNA replication is properly timed in recA mutants. We also find that more than 10% of recA mutant cells contain no DNA. These anucleate cells appear to arise from partitioning of all the DNA into one daughter cell and no DNA into the other daughter cell. Based on these and previously published results, we propose that RecA protein is required for equal partitioning of chromosomes into the two daughter cells.
