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DNA gyrase requirements distinguish the alternate pathways of Mu transposition
Tanya D Sokolsky1, Tania A Baker
1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Abstract:
The MuA transposase mediates transposition of bacteriophage Mu through two distinct mechanisms. The first integration event following infection occurs through a non-replicative mechanism. In contrast, during lytic growth, multiple rounds of replicative transposition amplify the phage genome. We have examined the influence of gyrase and DNA supercoiling on these two transposition pathways using both a gyrase-inhibiting drug and several distinct gyrase mutants. These experiments reveal that gyrase activity is not essential for integration; both lysogens and recombination intermediates are detected when gyrase is inhibited during Mu infection. In contrast, gyrase inhibition causes severe defects in replicative transposition. In two of the mutants, as well as in drug-treated cells, replicative transposition is almost completely blocked. Experiments probing for formation of MuA-DNA complexes in vivo reveal that this block occurs very early, during assembly of the transposase complex required for the catalytic steps of recombination. The findings establish that DNA structure-based signals are used differently for integrative and replicative transposition. We propose that transposase assembly, the committed step for recombination, has evolved to depend on different DNA /architectural signals to control the reaction outcome during these two distinct phases of the phage life cycle.
Insights
Bacteriophage Mu DNA transposition relies on MuA transposase. Gyrase is not essential for integration but is crucial for replicative transposition, impacting transposase complex assembly.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage Mu utilizes two distinct transposition mechanisms: non-replicative for integration and replicative for genome amplification during lytic growth.
- The MuA transposase is central to both transposition pathways.
- DNA supercoiling, influenced by gyrase, is a key factor in DNA structure and manipulation.
Purpose of the Study:
- To investigate the role of DNA gyrase and supercoiling in the two distinct transposition pathways of bacteriophage Mu.
- To determine how gyrase activity affects MuA transposase-mediated integration and replicative transposition.
Main Methods:
- Utilized a gyrase-inhibiting drug and various gyrase mutants to study bacteriophage Mu transposition.
- Assessed the formation of MuA-DNA complexes in vivo to pinpoint the stage of inhibition.
- Analyzed the outcomes of Mu infection under conditions of inhibited gyrase activity.
Main Results:
- Gyrase activity is dispensable for Mu integration, as evidenced by the formation of lysogens and recombination intermediates even when gyrase is inhibited.
- Replicative transposition is severely impaired by gyrase inhibition, with some mutants and drug treatments almost completely blocking this process.
- Inhibition of replicative transposition occurs early, during the assembly of the MuA transposase complex.
Conclusions:
- DNA structure signals are differentially employed by integrative and replicative transposition pathways of bacteriophage Mu.
- The assembly of the MuA transposase complex, a critical step for recombination, has evolved to sense distinct DNA architectural signals for controlling outcomes in different phage life cycle phases.