Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element.
We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling-circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross-complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha-helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin.
Wang
P Z
PZ
Department of Plasmid Biology, Public Health Research Institute of the City of New York, Inc., NY 10016.
Projan
S J
SJ
Henriquez
V
V
Novick
R P
RP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D012045:Regulatory Sequences, Nucleic Acid; D013211:Staphylococcus aureus / Q000235:genetics*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C043650:RepC protein, Staphylococcus aureus; C058757:RepD protein, Staphylococcus aureus