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A new domain of conjugative relaxase TrwC responsible for efficient oriT ‐specific recombination on minimal target sequences
Author(s) -
César Carolina Elvira,
Machón Cristina,
De La Cruz Fernando,
Llosa Matxalen
Publication year - 2006
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2006.05437.x
Subject(s) - recombinase , biology , recombination , site specific recombination , plasmid , dna , genetics , cre lox recombination , microbiology and biotechnology , gene , transgene , genetically modified mouse
Summary We show that relaxase TrwC promotes recombination between two directly repeated oriT s while related relaxases TraI of F and pKM101 do not. Efficient recombination required also relaxosome accessory protein TrwA even after deletion of TrwA binding sites at oriT , suggesting that the effect of TrwA is mediated by protein–protein interactions. TrwC relaxase domain was necessary but not sufficient to catalyse recombination efficiently. Full recombinase activity was obtained with the N‐terminal 600 residues of TrwC. The minimal target sequences required for recombination were different at each of the two involved oriT s: oriT1 could be reduced to the nic site and TrwC binding site, while oriT2 required an extended sequence including a set of iterons that are not required for conjugation. TrwC‐mediated integration of a transferred DNA into a resident oriT copy required a complete oriT in the recipient. We observed dramatic changes in the efficiency of recombination between tandem oriTs linked to the direction of plasmid replication and transcription through oriT1 . We propose that recombination is triggered by the generation of a single‐stranded DNA at oriT1 that causes TrwC nicking. The resulting TrwC‐DNA complex reacts with oriT2 , excising the intervening DNA. This intermediate can be resolved by host‐encoded replication functions.