Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element

• Published in: The EMBO journal Vol. 12; no. 1; pp. 45 - 52
• Main Authors: Wang, P.Z., Projan, S.J., Henriquez, V., Novick, R.P.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.01.1993
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Biological and medical sciences; Cloning, Molecular; DNA Replication; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Models, Structural; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Plasmids; Repetitive Sequences, Nucleic Acid; Replication; Replicon; Restriction Mapping; Sequence Homology, Amino Acid; Origin; Sequence specificity; Molecular structure; Replication initiation; DNA binding protein; Molecular interaction; Plasmid; Structure activity relation; Bacteria; Micrococcales; Micrococcaceae; Inverted repeated sequence; Staphylococcus aureus
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1993.tb05630.x

The leading strand replication origin of pT181 plasmids consists of two adjacent inverted repeat elements (IR‐II and IR‐III), which are involved in origin recognition by the initiator (Rep) protein. The conserved core element, IR‐II, which contains the initiation nick site, is induced by Rep to form a cruciform structure, probably the primary substrate for the initiation of rolling circle replication. The divergent repeat, IR‐III, constitutes the determinant of origin recognition specificity. We show here that the distal arm of IR‐III is not required for sequence‐specific recognition, whereas the proximal arm and central region are required. Since the initiator is dimeric, we presume that it binds symmetrically to IR‐III. A unique type of DNA‐protein interaction is proposed, in which the lack of sequence requirement for the distal arm is a consequence of binding to the adjacent IR‐II, which thereby polarizes the stringency of binding to the two arms of IR‐III. In addition, genetic evidence indicates that both the spacing and the phasing of IR‐II to IR‐III are crucial for function and that the central segment of IR‐III may serve to position the two flanking half‐sites for optimal interaction of Rep with IR‐III.