Challenging a paradigm: the role of DNA homology in tyrosine recombinase reactions

Abstract

A classical feature of the tyrosine recombinase family of proteins catalyzing site-specific recombination, as exemplified by the phage lambda integrase and the Cre and Flp recombinases, is the ability to recombine substrates sharing very limited DNA sequence identity. Decades of research have established the importance of this short stretch of identity within the core regions of the substrates. Since then, several new enzymes that challenge this paradigm have been discovered and require the role of sequence identity in site-specific recombination to be reconsidered. The integrases of the conjugative transposons such as Tn916, Tn1545, and CTnDOT recombine substrates with heterologous core sequences. The integrase of the mobilizable transposon NBU1 performs recombination more efficiently with certain core mismatches. The integration of CTX phage and capture of gene cassettes by integrons also occur by altered mechanisms. In these systems, recombination occurs between mismatched sequences by a single strand exchange. In this review, we discuss literature that led to the formulation of the current strand-swapping isomerization model for tyrosine recombinases. The review then focuses on recent developments on the recombinases that challenged the paradigm that was derived from the studies of early systems.

FIG. 1.

Substrates for tyrosine recombinases. (A) Phage lambda attP and attB sites have a 7-bp overlap region flanked by 7-bp inverted repeats (C, C′, B, and B′). P1, P2, and the P′123 sites are arm-type binding sites for Int. There are three binding sites for Xis, one for Fis (F), and three for IHF (H1, H2, and H′). (B) The minimal 34-bp loxP site. (C) The minimal 34-bp frt site. (D) Substrates for XerCD recombinases. The blue boxes are 11-bp binding sites for XerC or XerD as shown. The dif and psi sites have a 6-bp overlap region, while the cer site has an 8-bp overlap region. The cer and psi sites have accessory sequences for the accessory proteins (PepA and ArgR at cer and PepA and ArcA at psi). Vertical arrows are cleavage sites for the respective recombinases.

FIG. 2.

Recombination by tyrosine recombinases. Four recombinase monomers (blue ovals) bind to the substrates; two monomers are active (indicated by the tyrosine in position), and two are inactive. The active monomers cleave the first pair of DNA strands to form a 3′-phosphotyrosyl intermediate and free 5′-OH groups. Strand exchange results in an HJ intermediate. There is a conformational change, and the second pair of monomers becomes active, and they carry out the second set of DNA cleavages; the second round of strand exchanges and ligations results in the recombinant.

FIG. 3.

Integration of CTX phage into the V. cholerae dif1 site. The plus single-stranded phage DNA (purple) folds up into a hairpin structure, forming a target site with a central bulge for XerC and XerD recombinases. A single strand exchange carried out by XerC results in a branched structure that is resolved by replication or gap repair. The black lines indicate bases that arise through replication.

FIG. 4.

Model for CTnDOT excision and integration. D and D′ are core-type sites on CTnDOT (red), while B and B′ are core sites on the bacterial chromosome (purple). The 10-bp sequence 5′-GTAANTTTGC-3′ at one end of CTnDOT (D site) is also present in the target site (B site). During excision, IntDOT makes 7-bp staggered cuts (vertical arrows) at the ends of the integrated transposon. The element forms a covalently closed circular intermediate. A 5-bp region of heteroduplex is formed. The heteroduplex DNA is resolved after conjugation and complementary-strand synthesis in the recipient cell. IntDOT makes 7-bp staggered cuts on the double-stranded element and the attB site, and the element integrates into the recipient chromosome. The heteroduplex attL and attR sites are then resolved by DNA replication or repair in the recipient cell. The coupling sequences shown are a representative set of coupling sequences.

FIG. 5.

(A) The NBU1 attN1 and the Bacteroides attBT1-1 sites share a 14-bp region of identity (blue box). The pink box indicates the leu-tRNA gene. The direct (DR1, DR2, and DR3) and inverted (LIR and RIR) repeats in attN1 are probable binding sites for IntN1. The red box “N” indicates a second region of near identity. (B) Mutational analysis of the overlap region of NBU1. Vertical black arrows show cleavage sites for IntN1.