Analyzing DNA Replication Checkpoint in Budding Yeast
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.
Key wordsDNA replication checkpoint Mec1 Rad53 Kinase assay Budding yeast
We thank Dr. Susan M. Gasser for critical reading and helpful suggestions. Support was gratefully received from the Novartis Research Foundation, the Swiss Cancer League, and the FP7 Marie Curie Network, Imaging the DNA Damage Response (Image DDR).
- 22.Pellicioli A, Lucca C, Liberi G, Marini F, Lopes M, Plevani P, Romano A, Di Fiore PP, Foiani M (1999) Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase. EMBO J 18(22):6561–6572PubMedCentralPubMedCrossRefGoogle Scholar
- 27.Hegnauer AM, Hustedt N, Shimada K, Pike BL, Vogel M, Amsler P, Rubin SM, van Leeuwen F, Guenole A, van Attikum H, Thoma NH, Gasser SM (2012) An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks. EMBO J 31(18):3768–3783PubMedCentralPubMedCrossRefGoogle Scholar