Abstract

Serine integrases promote the recombination of two complementary DNA sequences, attP and attB, to create hybrid sequences, attL and attR. The reaction is unidirectional in the absence of an accessory protein called recombination directionality factor. We utilized tethered particle motion (TPM) experiments to investigate the reaction behaviors of two model serine integrases from Listeria innocua phage LI and Streptomyces coelicolor phage C31. Detailed kinetic analyses of wild-type and mutant proteins were carried out to verify the mechanisms of recombination directionality. In particular, we assessed the influence of a coiled-coil motif (CC) that is conserved in the C-terminal domain of serine integrases and is an important prerequisite for efficient recombination. Compared to wild type, we found that CC deletions in both serine integrases reduced the overall abundance of integrase (Int) att-site complexes and favored the formation of nonproductive complexes over recombination-competent complexes. Furthermore, the rate at which CC mutants formed productive synaptic complexes and disassembled aberrant nonproductive complexes was significantly reduced. It is notable that while the φC31 Int CC is essential for recombination, the LI Int CC plays an auxiliary role for recombination to stabilize protein-protein interactions and to control the directionality of the reaction.

Full Text

Published Version
Open DOI Link

Get access to 250M+ research papers

Discover from 40M+ Open access, 3M+ Pre-prints, 9.5M Topics and 32K+ Journals.

Sign Up Now! It's FREE

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call