Characterization of DNA helicases and nucleases from meiotic extracts of S. cerevisiae.

The formation of stable interactions between chromosomes of maternal and paternal origin-homologs-is required for their segregation during meiosis. To achieve this, cells take advantage of the recombination machinery, which promotes formation of reciprocal interhomolog exchanges, called crossovers, from the repair of self-inflicted DNA breaks. Important genetic studies led to the identification of key enzymes that control meiotic recombination. However, characterization of their biochemical properties when purified from meiotic cultures has been difficult to achieve. Here, we describe a simple approach to purify and characterize DNA repair enzymes from meiotic yeast cells. First, we provide a protocol to generate large-scale synchronous cultures. Second, we describe a general method to prepare meiotic extracts from which protein complexes can be immunoaffinity-purified. Finally, we detail how the purified material can be used for: (i) mass spectrometry-based analysis of interaction partners and posttranslational modifications, and (ii) monitoring enzymatic activities using synthetic DNA substrates.