Abstract
Site-specific recombinase Int mediates integration of the bacteriophage λ genome into the Escherichia coli chromosome. Integration occurs once the Int tetramer, assisted by the integration host factor IHF, forms the intasome, a higher order structure, within which Int, a heterobivalent protein, interacts with two nonhomologous DNA sequences: the core recombination sites and the accessory arm sites. The binding to these sites is mediated by the catalytic C-terminal domain (CTD) and the regulatory N-terminal domain (NTD) of Int, respectively. Within Int, the NTD can activate or inhibit the recombination activity of the CTD depending on whether the NTD is bound to the arm sites. The CTD alone cannot mediate recombination, and even when the NTD and the CTD are mixed together as individual polypeptides, the NTD cannot trigger recombination in the CTD. In this work, we set to determine what modifications can unlock the recombination activity in the CTD alone and how the CTD can be modified to respond to recombination-triggering signals from the NTD. For this, we performed a series of genetic analyses, which showed that a single mutation that stabilizes the CTD on DNA, E174K, allows the CTD to recombine the core DNA sequences. When the NTD is paired with the CTD (E174K) that also bears a short polypeptide from the C terminus of the NTD, the resulting binary Int can recombine arm-bearing substrates. Our results provide insights into the molecular basis of the regulation of the Int activity and suggest how binary recombinases of the integrase type can be engineered.