A proteomics approach to study the DNA damage checkpoint in Saccharomyces cerevisiae
- Author(s): Ponte de Albuquerque, Claudio
- et al.
The DNA damage checkpoint is a signal transduction pathway that is evolutionarily conserved from yeast to human. It is involved in the maintenance of the genome and regulation of cell cycle. Mutations in checkpoint proteins can lead to genomic instability and cancer in humans. This signal transduction pathway is composed by several kinases and adaptor proteins, and only a few substrates of these signaling transduction pathway are known. In Saccharomyces cerevisiae (budding yeast) Mec1 and Tel1 are the two upstream kinases, homologues to ATR and ATM in humans, involved in the initial sensing of the damage DNA or the stalled replication fork. Rad9 and Mrc1, orthologs of human 53BP1 and Claspin, are the main adaptor proteins of Mec1 and Tel1, and they activate the effector kinase Rad53 and Dun1. Recent technological advances in the field of proteomics have enabled us to identify and quantify thousands of peptides in a single experiment. In these studies I developed two separate multidimensional chromatography methods which when combined with quantitative proteomics can be used to examine the global changes in protein expression or phosphorylation. In Chapter 2, my first mutidimensional chromatography method was used to quantify changes in protein expression after DNA damage. In Chapter 3, a second mutidimensional chromatography method was developed, and it allowed an in- depth mapping of the phosphoproteome. Finally, we applied these methods to identify in vivo kinase substrates of the DNA damage checkpoint