The study of a rDNA replicator in Saccharomyces

Area of Science:

• * Molecular Biology
• * Yeast Genetics
• * Epigenetics

Background:

• * The centromeric plasmid Rcp-CEN3, which contains yeast ribosomal DNA (rDNA) autonomously replicating sequences (ARS), exhibits significant mitotic instability, with up to 50% loss per generation in most yeast strains.
• * Understanding the mechanisms behind this instability is crucial for plasmid maintenance and replication studies.

Purpose of the Study:

• * To elucidate the mechanisms underlying the high mitotic instability of the Rcp-CEN3 plasmid.
• * To investigate the role of autonomously replicating sequences (ARS) in centromeric plasmid stability.

Main Methods:

• * Construction of a modified Rcp-CEN3 plasmid featuring a tandem duplication of a rDNA ARS.
• * Comparative analysis of mitotic stability between mono-ARS and dual-ARS Rcp-CEN3 plasmids.
• * Genetic analysis to determine plasmid copy number and maintenance.

Main Results:

• * A tandem duplication of a rDNA ARS in Rcp-CEN3 markedly increased its mitotic stability compared to the mono-ARS version.
• * The observed mitotic stability of the dual-ARS plasmid aligned with the calculated stability of two independent mono-ARS plasmids.
• * Genetic analysis confirmed that both mono-ARS and dual-ARS plasmids are maintained in a single-copy state.

Conclusions:

• * The high mitotic instability of Rcp-CEN3 is linked to the lack of stringent replication control from the rDNA ARS.
• * Replication control mechanisms are critical for maintaining centromeric plasmid stability in yeast.
• * The findings provide insights into potential replication mechanisms of chromosomal rDNA repeats in yeast.