Improvement of recombination activity mediated by conjugative relaxases

Abstract

DNA processing during bacterial conjugation is driven by a nucleoprotein complex called relaxosome, where the relaxase is the key protein. Relaxases are site-and strand-specific endonucleases which recognize and cleave one strand of their cognate origin of transfer (oriT), and are transferred to the recipient cell covalently bound to the 5‘ end of the nicked DNA strand. TrwC is the relaxase of the conjugative plasmid R388. TrwC is also a site-specific recombinase capable of promoting efficient recombination between two cognate oriTs in the absence of conjugation (César et al., 2006). Moreover, it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell (Draper et al., 2005). TrwC is able to act as a recombinase and integrase usign as targets a discrete number of sequences, located in noncoding sites of the human genome (Agúndez et al., 2011; Agúndez et al., 2012). TraI is the relaxase of the F plasmid, highly related to TrwC, although it has been reported that TraI does not have recombinase activity (César et al., 2006). Using a TraI-mediated site-specific recombination assay we have demonstrated that TraI can catalyze recombination between two F-oriT copies, although the frequency of recombination mediated by F-TraI is very low compared to that of TrwC under similar assay conditions. This allows future search for TraI mutants with increased recombination activity, which will provide additional target sequences in the genomes of higher organisms. We have used random mutagenic PCR to mutagenize the trwC and traI genes, selecting for mutants with enhanced recombination activity. We have obtained a TrwC mutant with increased recombination activity, which is completely functional in conjugation. We have tested its recombinase activity in the presence or absence of TrwA, its accessory protein. Furthemore, we have tested its activity as a site-specific integrase in a bacterial assay. Mutants with enhaced recombination activity could be used for genomic engineering with better efficiency than wild-type proteins. These and other results show that bacterial conjugative relaxases could be used in the future as site-specific integrases in human cells, leading the way for the development of a novel system for genomic modification which could have applications in biotechnology and biomedicine.