Mechanisms of Activation and Inactivation of Rad53 Checkpoint Kinase

Fiorani, Simona (2008). Mechanisms of Activation and Inactivation of Rad53 Checkpoint Kinase. PhD thesis The Open University.



In many protein kinases, the phosphorylation of one or more residues in the activation segment is critical for driving the conformational change, which allows the activation of the kinase. The yeast checkpoint kinase Rad53 presents two phosphorylatable Threonine residues (T354 and T358) in the activation segment. In this thesis I have generated rad53 mutants with affected activation segment functions resulting from Alanine or Aspartate substitution of single or both the T354 and T358. These results pinpointed functional regulatory roles of the phosphorylations of both Threonine residues in the catalytic activation of Rad53. Interestingly, the rad53-T354D-T358D mutation caused the accumulation of the S129-phosphorylated isoform of the histone H2A even during an unperturbed cell cycle, thus indicating the accumulation of spontaneous DNA lesions. We further found that the accumulation of DNA lesions in rad53-T354D-T358D cells is due to high abundance of Sm11, that is the physiological inhibitor of the Ribonucleotide Reductase, thus suggesting that the main source of the DNA lesions in rad53-T354D-T358D cells is an inadequate pool of the DNA replication precursors dNTPs. In this thesis I also present the production and characterization of novel monoclonal antibodies directed against various isoforms of Rad53, including antibodies directed specifically to autophosphorylated and active Rad53.

In conclusion, the results and reagents described in this thesis may help to elucidate the essential role of the checkpoint kinase Rad53 in preserving genome integrity.

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