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The role of supercoiling in mycobacteriophage L5 integrative recombination
C E Peña1, J M Kahlenberg, G F Hatfull
1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Abstract:
The genome of temperate mycobacteriophage L5 integrates into the chromosomes of its hosts, including Mycobacterium smegmatis , Mycobacterium tuberculosis and bacille Calmette-Guérin. This integrase-mediated site-specific recombination reaction occurs between the phage attP site and the mycobacterial attB site and requires the mycobacterial integration host factor. Here we examine the role of supercoiling in this reaction and show that integration is stimulated by DNA supercoiling but that supercoiling of either the attP or the attB substrate enhances recombination. Supercoiling thus facilitates a post-synaptic recombination event. We also show that, while supercoiling is not required for the production of a recombinagenic intasome, a mutant attP DNA deficient in binding of the host factor acquires a dependence on supercoiling for intasome formation and recombination.
Insights
DNA supercoiling significantly enhances the integration of mycobacteriophage L5 into host chromosomes by facilitating a post-synaptic recombination event. This process is crucial for the phage
Area of Science:
- Molecular Biology
- Genetics
- Virology
Background:
- Mycobacteriophage L5 integrates its genome into the chromosomes of various mycobacterial hosts, including Mycobacterium tuberculosis.
- This integration is an integrase-mediated, site-specific recombination event occurring between phage attP and host attB sites, requiring mycobacterial integration host factor.
Purpose of the Study:
- To investigate the role of DNA supercoiling in the L5 phage integration process.
- To determine how supercoiling affects recombination at the attP and attB sites and intasome formation.
Main Methods:
- Examining the effect of DNA supercoiling on the site-specific recombination reaction between attP and attB.
- Analyzing intasome formation in the presence and absence of supercoiling, particularly with a mutant attP DNA.
Main Results:
- DNA supercoiling stimulates the integration of L5 phage into mycobacterial chromosomes.
- Supercoiling of either the attP or attB substrate enhances the recombination efficiency.
- While not essential for initial intasome production, supercoiling becomes critical for intasome formation and recombination in a mutant attP DNA deficient in host factor binding.
Conclusions:
- DNA supercoiling facilitates a post-synaptic stage of the L5 phage integration reaction.
- Supercoiling plays a significant role in overcoming DNA conformational barriers during recombination, especially when host factor binding is impaired.