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Volume 271, Number 22, Issue of May 31, 1996 pp. 13068-13076
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Mechanisms of Initiation and Termination Reactions in Conjugative DNA Processing
INDEPENDENCE OF TIGHT SUBSTRATE BINDING AND CATALYTIC ACTIVITY OF RELAXASE (TraI) OF IncP PLASMID RP4

(Received for publication, October 23, 1995, and in revised form, February 3, 1996)

From the Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, Dahlem, D-14195 Berlin, Federal Republic of Germany

The relaxase (TraI) of plasmid RP4 (IncP) plays a key role in initiation and termination of transfer DNA replication during conjugative transmission of the plasmid. TraI functions as a DNA strand transferase that cleaves a unique phosphodiester bond at nic of the transfer origin. The cleavage reaction consists in a reversible transesterification that leads to transfer of the 5 phosphoryl at nic to the hydroxyl group of TraI Tyr-22. Hence, cleavage results in the covalent attachment of TraI to the 5 terminus of the plasmid strand destined for transfer. To investigate the protein's ability to function in a ``second cleavage'' reaction proposed to terminate rolling circle mode transfer DNA replication, single-stranded oligonucleotides containing the nic region were immobilized at their 3 ends on magnetic beads and cleaved by TraI. The resulting covalent TraI-oligonucleotide adducts were active in the joining reaction but unable to cleave oligonucleotides containing an intact nic region, indicating that second cleavage probably requires a TraI dimer, since a monomer is insufficient. The covalently attached oligonucleotide determines the affinity of the relaxase for the 3 terminus of the T-strand. To further the biochemical characterization of TraI-catalyzed reactions, we used specific TraI mutants, showing that amino acid residues in each relaxase motif are involved in substrate binding. To uncouple substrate binding and cleaving-joining, we applied partially biotinylated TraI mutant proteins that were immobilized to magnetic beads. Using this approach we could demonstrate that tight DNA substrate binding and cleaving-joining are independent processes. Enhanced topoisomerase activity of some TraI mutants was correlated with low specific substrate binding affinity in conjunction with high cleaving-joining activity.

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