7.06_2004_PS4 - 7.06 Spring 2004 PS 4 1 of 10 Problem Set...

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Unformatted text preview: 7.06 Spring 2004 PS 4 1 of 10 Problem Set #4 Question 1. You are studying the DNA damage response pathway in a new species of yeast that your lab studies. In Particular, you are interested in learning the molecular mechanism behind cell cycle arrest due to DNA damage. You know that the DNA damage response causes an arrest in cell cycle progression and involves the cyclin dependent kinase CDK76. In vitro studies have shown that CDK76 is active when phosphorylated at 2 Tyr’s and is inactive when phosphorylated at a Thr and a Tyr. You suspect that DNA damage causes inactivation of CDK76 which leads to cell cycle arrest. Luckily you have CDK specific antibodies that recognize both the unphosphorylated form and the Thr and Tyr phosphorylated form. You decide to do a western blot with cells that have not been treated with a DNA damaging agent and cells that have been treated with a DNA damaging agent. a) Describe a step by step procedure for running a western blot for these two cell treatments. Below is the result of your western blot: 7.06 Spring 2004 Ladder CDK76 No DNA Damage PS 4 2 of 10 CDK76 W/ DNA Damage * Phosphorylated Thr and Tyr CDK76 § § * § Un-Phosphorylated CDK76 Now you have evidence that CDK76 is phosphorylated in response to DNA damage. Furthermore you suspect that an inhibitor is binding CDK76 so you want to find binding partners of both active and repressed phospho-CDK76. b) How would you go about finding intracellular binding partners of CDK76? Design a step by step experiment listing any controls you would include in your experiment. 7.06 Spring 2004 PS 4 3 of 10 Your screen picked up four potential binding partners of phospho-CDK76. Luckily these candidate binding partners have been previously described proteins, so you can purchase antibodies against each protein. You want more evidence that these proteins indeed bind CDK76 so you decide to IP (immuno precipitate) phospho-CDK76 using an antibody that specifically binds to all species of phosphorylated CDK76 (i.e. it will recognize both 2 Tyr and Thr and Tyr phosphorylated CDK76). So you IP cell lysates from yeast cells treated with a DNA damaging agent and from cells not treated with a DNA damaging agent. After you IP CDK76 you run a western with antibodies specific for the 4 candidate binding partners your expression cloning screen picked up. The results of this Co-IP are shown below: Ladder Protein W Protein X Protein Y No DNA Damage Protein Z Ladder Protein W Protein X Protein Y Protein Z DNA Damage c) Based on the data you have collected up to this point, what do these results suggest? 7.06 Spring 2004 PS 4 4 of 10 Luck is once again on your side because it just so happens that a mutant of protein Y exists. This protein Y mutant fails to arrest cell cycle progression in response to DNA damage. You decide to repeat your phospho-CDK76 Co-IP with wild-type cells and protein Y mutant cells treated with a DNA damaging agent and probed with an antibody against protein Y. The results of the Co-IP show the loss of the protein Y band in the mutant. d) With this data what do your results suggest? Now you want to know if cell cycle arrest is in fact due to loss of CDK76 activity. e) How would you test this? Design a detailed experiment. Question 2. While performing a mutagenesis screen designed to recover yeast mutants defective in the cell division cycle, you recover a cell cycle mutant that is temperature sensitive for growth at 37°C (it has a wild-type phenotype at 25°C). a. Describe two methods by which you can try to identify the cell cycle stage where this mutant may be arrested. b. After arresting the mutant with alpha factor and releasing the cells from the arrest at 25°C and 37°C, you collect samples at different time points for some FACS analysis. 7.06 Spring 2004 PS 4 5 of 10 Time after release from alpha factor 25°C 0 min 37°C 1C 2C 1C 2C 40 min 1C 1C 2C 2C 90 min 1C 2C 1C 2C 120 min 1C 2C 1C 2C What conclusions, if any, can you make about where in the cell cycle the mutant has arrested? 7.06 Spring 2004 PS 4 6 of 10 c. How could you more specifically identify the cell cycle stage at which the mutant is arrested? Why is this technique more informative than a FACS profile? d. You place an order for anti-tubulin antibodies from Acme Inc. so you can examine the spindle morphology of your mutants arrested at 37°C. Unfortunately, Pinky, the rat used to produce anti-tubulin antibodies has recently succumbed, creating a year-long back-log for anti-tubulin antibodies (no other companies produce this specific antibody). Can you think of another way by which one can examine whether cells are in G2, metaphase, or anaphase? Are there “molecular markers” (e.g. proteins that are produced, or degraded or morphological changes in the cell etc.) for each of these cell cycle stages that you can follow? e. You would like to identify the gene producing the arrest phenotype at 37°C. Describe in detail how you would do this. 7.06 Spring 2004 PS 4 7 of 10 f. After isolating and sequencing the gene that is responsible for inducing a cell cycle arrest at 37°C, you attempt to determine if it is similar to other cdc genes. To your surprise, this gene has not been identified in other screens for genes involved in the cell division cycle and so you name it cdc706. You believe the product of the CDC706 gene binds to and inhibits an inhibitor of the cell cycle, CdkI (Cdk1 functions by inhibiting Cdk2). Propose a reason to explain why cdc706 might arrest at 37°C? Describe in detail how you could test your model and include diagrams of experimental results that are consistent with your model. Be sure to include appropriate controls! 7.06 Spring 2004 PS 4 8 of 10 Question 3. You are studying the interaction between the Anaphase Promoting Complex (APC) and one of it’s specificity factors, Cdc20. In wild-type yeast, Cdc20 associates with the APC and promotes the polyubiquitination of Securin. Once polyubiquitinylated, Securin is degraded, liberating Separase to initiate anaphase. a) You are interested in identifying the domain of Cdc20 that provides the specificity necessary to target the APC specifically to Securin. You make 3 mutant strains, each with different domains of Cdc20 deleted and perform the following two experiments in each mutant: i) Immunoprecipitate Securin and immunoblot using an antibody that recognizes ubiquitin. ii) Immunoprecipitate the component of the APC that interacts with Cdc20 and immunoblot using a Cdc20 antibody. Your results are as follows: Wild Type Mut 1 Mut 2 Mut 3 Secuin IP, Ubiquitin Immunoblot Wild Type Mut 1 Mut 2 Mut 3 APC IP, Cdc20 Immunoblot Which Cdc20 mutant would you use for further study, and why? 7.06 Spring 2004 PS 4 9 of 10 b) Excited by these results, you quickly make a construct that expresses the wild type peptide which was deleted from the Cdc20 mutant that you selected above (let’s call this peptide A). When you overexpress peptide A in wild type yeast they arrest in metaphase. You perform a co-immunoprecipitation and obtain the following: IP: IB: Overexpression: Pep. A Pep. A Pep. B Securin Securin Securin Securin wild type Pep.A (no peptide) Pep. B Pep. C Pep. C (IB=immunoblot. The Wild type and Peptide A lanes are IP’s using antibodies to Peptide A. The Peptide B and Peptide C lanes are IP’s using antibodies for the regions of Cdc20 deleted in the other two mutants from Part 3A) Based on these results, how do you think peptide A helps target the Cdc20/APC to Securin? 7.06 Spring 2004 PS 4 10 of 10 c) How would you identify the exact region of peptide A responsible for its function? ...
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