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Journal of Bacteriology, April 2001, p. 2306-2315, Vol. 183, No. 7
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.7.2306-2315.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Functional Domains of Yeast Plasmid-Encoded Rep Proteins

A. Sengupta, K. Blomqvist,dagger A. J. Pickett, Y. Zhang, J. S. K. Chew, and M. J. Dobson*

Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Received 5 September 2000/Accepted 11 January 2001

Both of the Saccharomyces cerevisiae 2µm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2µm-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2µm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. Phone: (902) 494-7182. Fax: (902) 494-1355. E-mail: DOBSON{at}is.dal.ca.

dagger Present address: Department of Biotechnology, Royal Institute of Technology, S-100 44 Stockholm, Sweden.

Journal of Bacteriology, April 2001, p. 2306-2315, Vol. 183, No. 7
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.7.2306-2315.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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