SITE-SPECIFIC NICKING IN-VITRO AT ORI-T BY THE DNA RELAXASE OF TN5252

Tn5252 is a promiscuous streptococcal element capable of madiating hor izontal spread of multiple antibiotic resistance. To begin understandi ng the functional role of a transfer-related region in Tn5252, its nuc leotide sequence was determined. Sequence of this 3.3-kb DNA segment r evealed the presence of six open reading frames. The predicted amino a cid sequence of one of the open reading frames, ORF9, showed similarit y to a predicted protein product of the lactococcal conjugative plasmi d, pC1528. The deduced primary protein sequence of another, ORF4, show ed strong structural similarity to conserved regions of various prokar yotic DNA relaxases that initiate conjugal transfer by strand- and sit e-specific cleavage at the transfer origin. A hybrid protein containin g the ORF4 protein fused to the carboxyl terminal end of maltose bindi ng protein was purified from Escherichia coli and found to specificall y nick plasmids carrying a 2-kb DNA segment derived from the transposo n. The nicking reaction is protein concentration-dependent. These resu lts imply that the conjugative transposition of Tn5252 may involve rol ling circle replication and transfer of a unique DNA strand. (C) 1997 Academic Press.