The Mechanism of Dis2 Phosphorylation by Chk1 and Cell Cycle Dissertation

The Mechanism of Dis2 Phosphorylation by Chk1 and Cell Cycle Regulation - Dissertation Example PP1 and its role as a mitotic checkpoint xxxii 1.6. PP1 and cell cycle control xxxiv 1.7. Importance of regulatory subunits and their role in diseases xxxv 1.8. Human paralogues of Dis2 xli 1.9. Conclusion xliv Chapter 2 xlvii Materials and Methods xlvii 2.1. Preparation of media xlvii 2.2. Preparation of buffers xlviii 2.3. Preparation of stain l 2.4. Preparation of normal SDS-PAGE buffer and gels l 2.5. Preparation of PEMS solutions liii 2.6. Preparation of protein extracts for use in SDS-PAGE liv 2.7. Running of SDS-PAGE: lvii 2.8. Construction of yeast strains lviii 2.9. Preparation of membrane lx 2.10. Chk1-HA shift experiment lx 2.11. TCA protein extraction lxii 2.12. Immune localization of proteins in yeast cells lxiv 2.13. Drop test lxviii 2.14. Preparation of cells for imaging lxix 2.15. Acute cell survival lxxi Chapter 3 lxxiv Results lxxiv 3.1. Dephosphorylation of Chk1 at 40Â °C is not affected by Dis2 phosphatase lxxiv 3.2. Dephosphorylation of Hus1 at 40Â ° lxxviii 3. 3. Hypersensitivity to DNA damaging agent lxxxi 3.4. Structural changes to cells lxxxiv 3.5. Study on cell survival lxxxvi 3.6. Identification of hus1 isoforms xcii 3.7. Comparison of Dis2 with other proteins xciv Chapter 4 xcix Discussion xcix 4.1. Conclusion civ 5. Appendix cvi 5.1. Appendix – 1 Multiple sequence alignment of dis2 protein cvi 5.2. Appendix – 2 Alignment of dis2 from S. pombe with human protein serine/threonine phosphatase cviii 5.3. Appendix - 3 Significance sequence alignment of protein serine/threonine phosphatase-1 cxi Acknowledgment This thesis was made possible by the unrelenting support of my supervisors and peers. I thank the university and the department for providing me with the technical as well as educational support apart from laboratory facilities for carrying out this research. It has been a great pleasure to complete this thesis under the support and guidance of my professors. Hypothesis Chk1 kinase is phosphorylated at serine 345 in r esponse to DNA damage. Dis2 dephosphorylated this residue slowly when cells recover from a DNA damage-induced cell cycle arrest. A rise in temperature from 30Â °C to 40Â °C results in the rapid dephosphorylation of S345 by a yet unknown phosphatase. The purpose of this experiment is to investigate the requirement of Dis2 for the heat-induced phosphorylation and to investigate the cell cycle roles of this enzyme. Other phosphor-proteins such as Hus1 and Rad9 are also investigated. 1. Abstract Protein phosphatases are a group of enzymes which have very specific role in biological cell activities. Dis2 is a PP1 enzyme (serine-threonine phosphatase-1) which plays a key role in regulation of DNA damage signaling. Fission yeast Dis2 regulates the DNA damage respons by dephosphorylation of chk1 kinase at Ser 345. In eukaryotic cells, phosphorylation mainly occurs on three hydroxyl-containing amino acids, namely – serine, threonine, and tyrosine, of which serine is the predominant target. Dis2 dephosphorylates the DNA damage checkpoint kinase Chk1 (at Ser-345) to switch off the checkpoint signal. Interestingly, heat stress results in very rapid removal of the phosphate from Ser 345 by a yet unknown phosphatase. Given the requirement of Dis2 for the dephosphorylation of Ser 345 at the normal growth temperature of 30Â °C, this study was conducted to investigate the role of this phosphatase under heat stress condition modification of Ser 345 is easily detected as a band shift of the protein which changes from a closed, low activity conformation