Abstract
Tyrosine (Y)-recombinases have evolved to deliver mechanistically different reactions on a variety of substrates, but these evolutionary transitions are poorly understood. Among them, integron integrases are hybrid systems recombining single- and double-stranded DNA partners. These reactions are asymmetric and need a replicative resolution pathway, an exception to the canonical second strand exchange model of Y-recombinases. Integron integrases possess a specific domain for this specialized pathway. Here we show that despite this, integrases are still capable of efficiently operating the ancestral second strand exchange in symmetrical reactions between double-stranded substrates. During these reactions, both strands are reactive and Holliday junction resolution can follow either pathway. A novel deep-sequencing approach allows mapping of the crossover point for the second strand exchange. The persistence of the ancestral activity in integrases illustrates their robustness and shows that innovation towards new recombination substrates and resolution pathways was a smooth evolutionary process.
Highlights
Tyrosine (Y)-recombinases have evolved to deliver mechanistically different reactions on a variety of substrates, but these evolutionary transitions are poorly understood
The recombination reaction is a stepwise process that starts with the cleavage and transfer of one strand, leading to the transient formation of a Holliday junction (HJ) that is further resolved through a second exchange of strands
During attC site folding, the imperfect pairing of both arms of the bs allows for the protrusion of a set of extrahelical bases (EHBs) (Fig. 1d) that are key in strand selectivity, favouring the recombination of the bs[18,19]
Summary
For this work we chose to study attI1, the integration site of the most widespread and best-studied integron platform, the Class 1 integron. To test the hypothesis that the attI1 Â attI1 reaction can take place through both the bottom and the top strand, we used an attI1 carried on a mismatched covalent circle[21] These synthetic circles mimic non-replicative plasmids assembled using complementary strands produced separately and hybridized together in vitro. This allowed us to tag each strand with a mutation in a restriction site (SacII or NarI) so that the restriction pattern of cointegrates reveals which strand is transferred during recombination (Fig. 2a). The mismatch-containing region was PCR amplified from 100 recombinant clones and used for the restriction pattern analysis (Fig. 2c), revealing that both strands of a oriP15a pSU-attI1
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