NICKING BY TRANSESTERIFICATION - THE REACTION CATALYZED BY A RELAXASE

DNA relaxases play an essential role in the initiation and termination of conjugative DNA transfer, Purification and characterization of rel axases from several plasmids has revealed the reaction mechanism: rela xases nick duplex DNA in a site-and strand-specific manner by catalysi ng a transesterification. The product of the reaction is a nicked doub le-stranded DNA molecule with a sequestered 3'-OH and the relaxase cov alently bound to the 5' end of the cleaved strand via a phosphotyrosyl linkage. The relaxase-catalysed transesterification is isoenergetic a nd reversible; a second transesterification ligates the nicked DNA. Ho wever, the covalent nucleoprotein complex is relatively long-lived, a property that is likely to be essential for its role as an intermediat e in the process of conjugative DNA transfer, Subsequent unwinding of the nicked DNA intermediate is required to produce the single strand o f DNA transferred to the recipient cell. This reaction is catalysed by a DNA helicase, an activity intrinsic to the relaxase protein in some , but not all, plasmid systems, The first relaxase-catalysed transeste rification is essential for initiation of conjugative strand transfer, whereas the second is presumably required for termination of the proc ess. The relaxase, in conjunction with several auxiliary proteins, for ms the relaxation complex or relaxosome first described nearly 30 year s ago as being associated with conjugative and mobilizable plasmids.