Functional domains of yeast plasmid-encoded Rep proteins

Area of Science:

• Molecular and Cellular Biology
• Yeast Genetics
• Epigenetics and Non-Mendelian Inheritance

Background:

• The 2-micrometer circle is a high-copy number plasmid in Saccharomyces cerevisiae.
• Efficient distribution of the 2-micrometer circle to daughter cells relies on the plasmid-encoded Rep1 and Rep2 proteins.
• Understanding the molecular mechanisms of Rep protein function is key to comprehending plasmid stability.

Purpose of the Study:

• To elucidate the interaction domains of Rep1 and Rep2 proteins.
• To investigate the roles of these domains in protein self-association and interaction.
• To propose a model for the mechanism of 2-micrometer circle plasmid segregation.

Main Methods:

• Two-hybrid assays to detect protein-protein interactions.
• In vitro protein interaction and baiting assays.
• Southwestern analysis to determine DNA-binding activity.
• Complementation assays using truncated Rep1 fusion proteins.

Main Results:

• The N-terminal 129 amino acids of Rep1 mediate self-association and Rep2 interaction.
• Distinct but overlapping domains in Rep1 are responsible for self-association and Rep2 interaction.
• Rep2 possesses two non-overlapping domains for self-association; its N-terminal domain interacts with Rep1, and its C-terminal domain binds DNA.

Conclusions:

• Rep1 and Rep2 proteins likely polymerize on the plasmid stability locus, forming a segregation complex.
• The interplay between Rep1 and Rep2 interactions, along with DNA binding by Rep2, facilitates plasmid segregation.
• Competition for Rep1 association regulates the assembly and disassembly of the segregation machinery.