Purification and properties of Int-h, a variant protein involved in site-specific recombination of bacteriophage lambda.

The bacteriophage lambda integrase catalytic domain can be modified to act with the regulatory domain as a recombination-competent binary recombinase

“…Next, we tested whether a mutation that can potentially stabilize the CTD on DNA could help trigger the recombination activity of the CTD. For this, we first tried the int-h mutation, E174K, which was shown to relax the requirement for the integration host factor (IHF) during recombination and to increase the binding affinity of Int ( 9 , 10 , 11 ). We thus added the int-h mutation to C65 and C75 and tested these modified CTD variants, C65(E174K) and C75(E174K), respectively, using the positive reporter vector that contained the attP / attB substrate pair.…”

“…1 and 2 ). Int with the int-h mutation was shown to perform recombination without the accessory protein IHF, which bends DNA within attP , thus helping Int to assemble the intasome and to have increased binding affinity for the recombination targets ( 10 , 11 ). The other mutation that we tested, E218K ( 13 ), which was suggested to increase the overall activity of Int and possibly its binding affinity, was not able to stimulate the recombination activity of C65 if added alone but was able to enhance the activity of C65(E174K), Figure 2 D .…”

“…To answer these questions, we performed a series of genetic analyses that utilized the reporter vectors with different recombination substrate pairs and the vectors that expressed variants of the NTD and the CTD. We found that a single modification that stabilizes the CTD on DNA is sufficient to allow this domain to recombine the core DNA sequences: the int-h mutation, E174K ( 9 , 10 , 11 ). We also found that when the NTD is paired with the CTD that has the int-h mutation and a short polypeptide from the C terminus of the NTD, the resulting binary Int can recombine several arm site–bearing substrates.…”

λ-integrase mediated seamless vector transgenesis platform

“…Studies have shown that wild-type integrase requires DNA supercoiling of target sites to catalyze recombination (167,377). However, the mutant Int variant, Int-h/218 can perform both integrative and excisive recombination in the absence of negative DNA supercoiling in vitro (173,178,378). This might be one of the reasons for efficient recombination by Int-h/218 on a linearized plasmid in the absence of any DNA supercoiling.…”