A Method for Physical Analysis of Recombination Intermediates in Saccharomyces cerevisiae.

Abstract

Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions (SEIs) and double-Holliday junctions (dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication.