Requirements for catalysis in the Cre recombinase active site

Abstract

Members of the tyrosine recombinase (YR) family of site-specific recombinases catalyze DNA rearrangements using phosphoryl transfer chemistry that is identical to that used by the type IB topoisomerases (TopIBs). To better understand the requirements for YR catalysis and the relationship between the YRs and the TopIBs, we have analyzed the in vivo and in vitro recombination activities of all substitutions of the seven active site residues in Cre recombinase. We have also determined the structure of a vanadate transition state mimic for the Cre-loxP reaction that facilitates interpretation of mutant activities and allows for a comparison with similar structures from the related topoisomerases. We find that active site residues shared by the TopIBs are most sensitive to substitution. Only two, the tyrosine nucleophile and a conserved lysine residue that activates the 5'-hydroxyl leaving group, are strictly required to achieve >5% of wild-type activity. The two conserved arginine residues each tolerate one substitution that results in modest recombination activity and the remaining three active site positions can be substituted with several alternative amino acids while retaining a significant amount of activity. The results are discussed in the context of YR and TopIB structural models and data from related YR systems.

Figure 1.

Tyrosine recombinases and type IB topoisomerases. (A) Tyrosine recombinase strand-exchange mechanism to generate a HJ intermediate. The HJ intermediate can be converted to starting substrates by exchange of the same strands or to recombinant products by exchange of the second pair of strands by the remaining two protein subunits. (B) The cleave–rotate–ligate mechanism of supercoil relaxation used by type IB topoisomerases. The two enzyme families use nearly identical mechanisms to catalyze the phosphoryl transfer reactions.

Figure 2.

Structure of Cre–DNA–vanadate transition state mimic. (A) Sequence of the DNA substrate used, with vanadium incorporation indicated by ‘v’. The 14-bp recombinase binding elements that surround the central 6-bp crossover region are shaded. The ‘+1’ and ‘–1’ base pair labels are used to facilitate comparison with related TopIB structures. (B) Weighted 2F_o–F_c electron density following refinement at 2.3 Å resolution. The map is contoured at 1.2σ. Glu176 and His289 are not visible in this view. (C) Overall structure of the synaptic complex, viewed from the N-terminal domains of the recombinase subunits. The activated subunits containing vanadate linkages are colored green. (D) View of a single Cre subunit bound to a loxP half-site within the tetrameric complex, rotated from the boxed subunit in (C). The tyrosine linkage to vanadate is circled.

Figure 3.

The Cre–DNA–vanadate transition state mimic active site. (A) Stereo view of the ‘activated’ site where vanadate has been incorporated. (B) Schematic of the activated site with additional residues and hydrogen bonding interactions indicated. Well-ordered water molecules are drawn as red spheres in (A) and (B).

Figure 4.

In vivo recombination activities of Cre active site mutants. (A) F' reporter used to score in vivo activity. If Cre excises a transcriptional terminator located downstream of the promoter, the streptomycin resistance and LacZYA gene products are expressed. (B) in vivo recombination results for Cre active site substitutions. Activity definitions: 0: no activity observed; +: <5% wild-type, ++: 5–50% wild-type, +++: >50% wild-type.

Figure 5.

In vitro recombination activities of Cre active site mutants. (A) Schematic of the excision assay. (B) Native PAGE of in vitro recombination reactions following protein digestion for the H289 panel of substitutions. In addition to the 602 bp substrate (labeled ‘S’) and the 234 bp product (labeled ‘P’), a band corresponding to a covalent Cre–DNA intermediate can be observed migrating slightly above the product band for some mutants. This is a nicked 234-bp covalent 3′-phosphotyrosine adduct. The corresponding covalent intermediate associated with the substrate is not well resolved. Identification of covalent and HJ intermediate bands are described in more detail in Supplementary Figure S2. (C) In vitro recombination results. Activity definitions: 0: no activity observed; +: <5% wild-type, ++: 5-50% wild-type, +++: >50% wild-type.