Abstract
The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.
Highlights
Apl is one of a large family of recombination directionality factors (RDFs) (Lewis and Hatfull, 2001), that modulate the directionality of site-specific recombination reactions catalysed by the tyrosine integrase/recombinase proteins (Esposito and Scocca, 1997)
RDFs are small proteins that bind to the DNA flanking the recombination site and, by altering the DNA architecture or by interacting with the integrase protein, act to alter the binding of the integrase complex to promote excision of the prophage
Strains were grown at 37°C in lysogeny broth (LB), with the addition of ampicillin (100 μg ml-1 for pZE15 based plasmids) and kanamycin (50 μg ml ml-1 for pUHA1) where necessary. pZE15-Plac-LacZ was constructed by inserting the lacZ gene into the BamHI and Hind-III sites of the ampicillin resistant, colE1 based plasmid pZE15 (Dodd et al, 2001)
Summary
Apl is one of a large family of recombination directionality factors (RDFs) (Lewis and Hatfull, 2001), that modulate the directionality of site-specific recombination reactions catalysed by the tyrosine integrase/recombinase proteins (Esposito and Scocca, 1997). RDFs appear to share a mode of DNA binding in which protomers bind with high cooperativity in a head-to-tail manner to tandem DNA repeats spaced one DNA turn apart, shown for λ Xis (Sam et al, 2002), Gifsy-1 Xis (Flanigan and Gardner, 2007) and Pukovnik Xis (Singh et al, 2014). RDFs have been shown to cause large bends in attachment site (att) DNA (λ Xis (Thompson and Landy, 1988) (Cho, Gumport and Gardner, 2002), P2 Cox (Ahlgren-Berg et al, 2009), L5 Xis (Lewis and Hatfull, 2003), P4 Vis (Calì et al, 2004), W Cox (Ahlgren-Berg et al, 2009), P22 Xis (Mattis, Gumport and Gardner, 2008) and Pukovnik Xis (Singh et al, 2014)).
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