Functional domains of yeast plasmid-encoded Rep proteins.
Both of the Saccharomyces cerevisiae 2 microm 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 2 microm-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 2 microm 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.
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
A
A
Sengupta
K
K
Blomqvist
A J
AJ
Pickett
Y
Y
Zhang
J S
JS
Chew
M J
MJ
Dobson
D004247:DNA / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000502:physiology; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D005656:Fungal Proteins; C051237:REP protein, yeast plasmid; D011993:Recombinant Fusion Proteins; D004247:DNA