High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast.

Area of Science:

• Molecular Biology
• Genetics
• Yeast Biology

Background:

• DNA replication origins are crucial for genome duplication and efficient plasmid maintenance in expression systems.
• The yeast autonomously replicating sequence (ARS) assay is vital for studying replication origins, but data is limited to a few species.
• Limited knowledge of ARS in diverse yeasts hinders understanding of origin function and evolution.

Purpose of the Study:

• To develop rapid, sequencing-based methods for comprehensive mapping and characterization of ARSs in yeast genomes.
• To define the molecular determinants of ARS function at single-nucleotide resolution using deep mutational scanning.
• To design short, synthetic DNA sequences with maximal ARS function.

Main Methods:

• Developed a sequencing-based suite of methods for mapping genomic inserts supporting plasmid replication.
• Employed massively parallel deep mutational scanning to analyze ARS function.
• Utilized high-throughput sequencing for single-nucleotide resolution analysis of ARS determinants.

Main Results:

• Successfully mapped and characterized ARSs within a yeast genome with high resolution.
• Identified key molecular determinants governing ARS function.
• Designed synthetic DNA sequences that retain significant ARS activity.

Conclusions:

• The developed methods enable rapid and comprehensive study of ARS function in diverse yeast species.
• This work provides insights into ARS structure and function, facilitating the design of synthetic origins.
• The approach can be broadly applied to understand and engineer ARS function across various biological systems.