Control of directionality in L5 integrase-mediated site-specific recombination.
Mycobacteriophage L5 is a temperate phage that forms lysogens in Mycobacterium smegmatis. These lysogens carry an integrated L5 prophage inserted at a specific chromosomal location and undergo subsequent excision during induction of lytic growth. Both the integrative and excisive site-specific recombination events are catalyzed by the phage-encoded tyrosine integrase (Int-L5) and require the host-encoded protein, mIHF. The directionality of these recombination events is determined by a second phage-encoded protein, Excise, the product of gene 36 (Xis-L5); integration occurs efficiently in the absence of Xis-L5 while excision is dependent upon it. We show here that Xis-L5 binds to attR DNA, introduces a DNA bend, and facilitates the formation of an intasome-R complex. This complex, which requires mIHF, Xis-L5 and Int-L5, readily recombines with a second intasome formed by Int-L5, mIHF and attL DNA (intasome-L) to generate the attP and attB products of excision. Xis-L5 also strongly inhibits Int-L5-mediated integrative recombination but does not prevent either the protein-DNA interactions that form the attP intasome (intasome-P) or the capture of attB, but acts later in the reaction presumably by preventing the formation of a recombinagenic synaptic intermediate. The mechanism of action of Xis-L5 appears to be purely architectural, influencing the assembly of protein-DNA structures solely through its DNA-binding and DNA-bending properties.
Lewis
John A
JA
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Hatfull
Graham F
GF
D001426:Bacterial Proteins; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D019426:Integrases / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011995:Recombination, Genetic / Q000502:physiology*
C108089:AttR protein, bacteria; D001426:Bacterial Proteins; D008565:Membrane Proteins; D004247:DNA; D019426:Integrases