Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis

Area of Science:

• * Molecular Biology
• * Yeast Genetics
• * DNA Replication

Background:

• * Autonomous replicating sequences (ARS) are crucial for DNA replication initiation in eukaryotes.
• * Understanding ARS function in different yeast species provides insights into conserved replication mechanisms.

Purpose of the Study:

• * To analyze the replication activity of the Kluyveromyces lactis chromosomal ARS, KARS101.
• * To identify and characterize the functional elements within KARS101 essential for its activity.
• * To investigate the binding of nuclear proteins to KARS101 and compare its function in Saccharomyces cerevisiae.

Main Methods:

• * Analysis of replication intermediates.
• * Deletional analysis of KARS101.
• * Gel shift assays and in vitro footprinting using yeast nuclear extracts.
• * Functional assays in Saccharomyces cerevisiae.

Main Results:

• * KARS101 functions as a chromosomal replicator in Kluyveromyces lactis.
• * A conserved 50 bp sequence within KARS101 is essential for its replicator activity in both plasmids and chromosomes.
• * This conserved sequence binds Kluyveromyces lactis nuclear proteins in an ATP-dependent manner, requiring a 40 bp core for efficient binding.
• * KARS101 also functions as an ARS in Saccharomyces cerevisiae, with a conserved dodecanucleotide sequence essential for binding and activity.

Conclusions:

• * A conserved sequence element is critical for ARS function in Kluyveromyces lactis.
• * The binding of nuclear proteins to this element is essential for ARS activity and resembles aspects of the origin recognition complex.
• * KARS101 demonstrates functional conservation of ARS elements between Kluyveromyces lactis and Saccharomyces cerevisiae.