| The DNA damage checkpoint response detects DNA damage and responds to the damage by promoting DNA repair, transcriptional regulation, and cell cycle arrest. Prior to the beginning of this dissertation the checkpoint sensor proteins in S. cerevisiae were identified as Ddc1, Mec3, Rad9, Rad17 and Rad24. However, none of the sensors had been shown to bind DNA directly, an anticipated function of checkpoint sensors. To characterize these proteins a biochemical approach was taken to test whether any of the checkpoint sensor proteins could detect DNA.; The associated DNA binding properties of Rad24 and Rad9 were identified and characterized for the first time. Both of these checkpoint sensor proteins have an affinity for ssDNA, a common intermediate DNA structure of most DNA repair processes. In addition, the DNA damage checkpoint mutant protein Rad24-1 is defective for binding to ssDNA, suggesting that Rad24 DNA binding is required for its function in the checkpoint response.; The potential exonuclease activity of Rad 17 tested using purified protein and various DNA substrates. This study was based on reports that the Rad 17 homolog Rec 1 from U. maydis is a 3′→5 ′ DNA exonuclease, and genetic data that indicated that Rad 17 has a role in telomere degradation. Exonuclease assays with Rad17 protein preparations and ssDNA found an associated weak exonuclease that was not significantly above background levels.; Conserved residues of Rad 17 thought to be required for exonuclease activity and checkpoint activity were mutated and studied for their affect on the DNA damage checkpoint. These studies imply that in addition to the region of Rad17 that is homologous to PCNA, the long carboxy-terminal region of Rad17 is also required for its checkpoint activity.; Collectively, these studies suggest that the common DNA repair intermediate structure single-stranded DNA is recognized by multiple checkpoint sensor proteins to initiate the DNA damage checkpoint response. This suggests that the initiation of the checkpoint response is the recognition of a single DNA structure instead of the many different structures of primary DNA damage by free radicals, UV, γ-radiation, alklylation, double strand breaks, and base mismatches. |
