Analyzing DNA Replication Checkpoint in Budding Yeast

Abstract

Checkpoints are conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. Cells also possess a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication fork progression. In the budding yeast Saccharomyces cerevisiae, the DNA replication checkpoint controls the timing of S-phase events such as origin firing and spindle elongation. This checkpoint also upregulates dNTP pools and maintains the replication fork structure in order to resume DNA replication after replication block. Many replication checkpoint factors have been found to be tumor suppressors, highlighting the importance of this checkpoint pathway in human health. Here we describe a series of protocols to analyze the DNA replication checkpoint in S. cerevisiae.

Yeast PP4 Interacts with ATR Homolog Ddc2-Mec1 and Regulates Checkpoint Signaling

“…Enzymatic assays, recovery and drop assays, fluorescence activated cell sorting (FACS) analysis, and Rad53 and H2A phosphorylation analysis were done as described previously (Hustedt and Shimada, 2014) or as detailed in Supplemental Experimental Procedures. AntiGFP IP was carried out as described for kinase assays, except that the lysis buffer was supplemented with protease and phosphatase inhibitors (see Supplemental Experimental Procedures) and bead-bound protein complexes were washed three times with lysis buffer prior elution with 0.2 M glycine.…”