The genomes of all living organisms are constantly damaged by exposure to harmful radiation, such as x-rays, -rays and UV. Pathways responsible for surveillance of genomic integrity have been termed DNA checkpoints. Genetic studies in yeast have identified several genes important for DNA damage-dependent checkpoints such as: RAD9, RAD53, Mec1, RAD17, RAD24 and DDC1. In spite of the discovery of these genes and their influence on down stream effects on the checkpoint pathways, biochemical information relating to the encoded products has been lacking. There is converging evidence to suggest that the checkpoints may work by tightly controlling the dNTP pools during DNA damage. A yeast based genetic study has identified a protein called Sml1 which is thought to be a negative regulator of ribonucleotide reductase 1 (RNR 1), a key enzyme in dNTP synthesis. During DNA damage or the S phase of cell cycle, the Mec1/Rad53/Dun1 cascade controls Sml1. The key regulatory event involves Sml1 phosphorylation, which results in initiating its degradation after DNA damage or upon entering the S phase. A recent study has identified the kinase responsible for Sml1 phosphorylation as Dun1, a nuclear protein that also controls ribonucleotide reductase (RNR) transcription. Almost all of the published data characterizing the Sml1-Rnr1 interactions come from genetic screens (Zhao et al., 1998; 2001,2002). However, very little is known about Sml1-Rnr1 interactions at the molecular level. The proposed model of the inhibition of dNTP synthesis involves unphosphorytated Sml1 binding Rnr1 and blocking the RNR activity. This proposal will address the following three key areas relating to the biochemistry of Sml1. Firstly, what are the phosphorylation sites of Sml1, and what impact will phosphorylation have on the Sml1 structure, stability and degradation. Secondly, analyze the interactions made by the unphosphorylated Sml1 with Rnr1. Finally, the long-term goal of this proposal is to solve the three-dimensional structures of Sml1 and the Sml1-Rnr1 complex. These questions will be addressed by using an integrated approach employing state-of-the-art structural biological technologies in mass spectrometry, biochemistry and x-ray crystallography.
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