Bacteriophage P1 site-specific recombination: III. Strand exchange during recombination at lox sites

doi:10.1016/0022-2836(81)90384-3

Journal of Molecular Biology

Volume 150, Issue 4, 25 August 1981, Pages 603-608

https://doi.org/10.1016/0022-2836(81)90384-3 Get rights and content

Abstract

When hybrid λ-P1 phages containing either loxP or loxR sites are crossed under conditions where only the P1 lox site-specific recombination system is active, most of the crossovers occur in a region of the DNA that contains the lox sites. The remainder of the lox-mediated crossovers (4% in a P × P cross and 32% in a P × R cross) occur close to, but outside of, either loxP or loxR. These latter crossovers are not detected if one of the partners in the cross carries a deletion of loxP. We explain these results by an exchange of strands at lox sites and a migration of the resulting cross-strand junction outside of lox.

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Bacteriophage P1 Cre protein is a member of the tyrosine recombinase/topoisomerase 1B family of DNA strand exchange proteins.1–4 Tyrosine recombinases are characterized by a common chemical mechanism and a shared active-site structure that includes a tyrosine nucleophile and an Arg-His-Arg “catalytic triad” (Figure 1(a) and (b)).3,5–8 Recombination requires only Cre and substrate DNA target site, LoxP (Figure 1(c)).9–11
During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8 bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5′ (S1 nucleotide) or 3′ (S1′ nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1′ substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the “conformational switch” isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289 may be to selectively stabilize the “activated” phosphate conformation in order to promote cleavage.

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^☆: Research sponsored by the National Cancer Institute under contract no. NO1-CO-75380 with Litton Bionetics.

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Journal of Molecular Biology

Letter to the editorBacteriophage P1 site-specific recombination: III. Strand exchange during recombination at lox sites☆

Abstract

J. Mol. Biol

J. Mol. Biol

Genetics

Genetics

J. Mol. Biol

Letter to the editor
Bacteriophage P1 site-specific recombination: III. Strand exchange during recombination at lox sites☆