Dohrmann, P.R., G. Oshiro, M. Tecklenburg, & R.A. Sclafani (1999) RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae. Genetics 151: 965-977.
The eukaryotic cell cycle is a highly regulated process requiring dozens of proteins to coordinate the series of complex events, sometimes occurring in parallel, which must proceed in an orderly manner. After years of studying the genes encoding these regulators, Hartwell and coworkers realized that a second class of regulators existed whose function is to scrutinize the proper completion of each major event in the cell cycle. Hartwell termed these surveillance functions 'checkpoints' (reviewed in Hartwell & Weinert, 1989; Weinert, 1998; Russell, 1998; Lew, 2000, and elsewhere). The events that are monitored by checkpoint pathways include the completion of DNA replication, attachment of kinetochores to the spindle, sister chromatid separation, and cytokinesis.
Progress from one phase of the cell cycle to the next is blocked by the appropriate checkpoint pathway, which becomes activated when the events of the previous cell cycle phase are not properly completed. The checkpoint pathways ensure that DNA replication occurs only once per cell cycle and that replication alternates with sister chromatid segregation and cytokinesis. A checkpoint pathway includes sensor functions that detect whether or not the cell cycle event has been completed, transducer functions that respond to the sensor and act on target functions which block exit from that stage of the cell cycle. The checkpoint pathway thereby provides a delay that hopefully allows for the completion of the necessary steps.
RAD53 encodes a protein kinase involved in all three of the DNA damage checkpoints (reviewed in Weinert, 1998). Other evidence suggests that Rad53
protein kinase is also required for the initiation of DNA replication and Article 17
addresses this role of Rad53p.
1. Summarize the results of previous published studies that suggest a role for Rad53p in the initiation of DNA replication distinct from its checkpoint function.
2. Describe the screen used to identify the Isd (lethal with seven defect) mutations.
(a) Give the name and genotype of the strain used to isolate the Isd mutations.
(b) Describe the red/white adenine colony sectoring assay.
(c) In your own words, describe why a colony carrying a mutation in a gene other than CDC7 that is synthetically lethal with cdc7-l will not have red sectors.
(d) How many nonsectoring clones were obtained?
(e) All nonsectoring clones carried recessive alterations based on the cross to PDY093. Diagram the cross including the cdc7 and Isd genotypes and sectoring phenotypes of both parental strains and describe the sectoring phenotype of the diploid. Explain why the phenotype of the diploid indicates that the Isd mutation is recessive.
(f) If one of the mutations had been dominant, what would have been the phenotoype of the diploid?
3. Eighteen of the mutants showed single gene segregation when crossed to PDY093. These were placed into complementation groups.
(a) Mutants 18 and 24 are in the same complementation group, LSD6. Diagram the cross between these mutants that demonstrates that they are alleles of LSD6. Show the genotypes of the parental strains with regard to LSD6 and CDC7, and ADE2 and plasmid pADE2. Give the sectoring phenotype of the parents and the diploid.
(b) Mutants 6 and 18 are in different complementation groups. What is the sectoring phenotype of the diploid heterozygous for these mutant genes?
4. Describe the cloning of LSD1. Include the genotype of the strain used to construct the library, the genotype of the host strain transformed with the library, and the phenotype used to identify the clone containing the plasmid-borne LSD1.
5. How did the authors demonstrate the LSD1 was RAD53? (Two methods.)
6. Describe the methods used to demonstrate that the checkpoint function of Rad53p is not affected in the rad53-3J allele.
7. Which model of enhancement best describes the genetic interaction between mutations in CDC7 and RAD531 Why? (Use the results in Table 3 for your answer.)
8. Based in part on the results in Table 4, the authors conclude that bobl-1 does not suppress rad53-31 as it does cdc7-l and db/4 mutations. What interfering phenomenon complicated their analysis of these results?
9. The results of two-hybrid analysis shown in Figure 2 indicate that Rad53p and Dbf4 interact.
(a) Approximately which residues of Rad53p are involved in the interaction?
(b) The strain carrying pRad53iDB and pDbf4AD gives 15 Miller units of ¡3-galactosidase activity. The authors conclude that there is no interaction. Why?
10. The results in Figures 3 and 4 indicate that DBF4 expression at the level of transcription and translation is dependent on Rad53p.
(a) These results do not explain the synthetic lethality of rad53-31 with cdc7-1 and dbf4-l. Why?
(b) What explanation do the authors provide?
(c) Which of the results presented in Figures 3 and 4 is consistent with this interpretation? (List at least two.)
Hartwell, L. & T.A. Weinert (1989) Checkpoints: controls that ensure the order of cell cycle events. Science 246: 629-634. Lew, D. (2000) Cell-cycle checkpoints that ensure coordination between nuclear and cytoplasmic events in Saccharomyces cerevisiae. Curr. Opin. Genet. Dev. 10: 47-53. Russell, P. (1998) Checkpoints on the road to mitosis. Trends Biochem. Sci. 23: 399-402. Weinert, T. (1998) DNA damage checkpoints update: getting molecular. Curr. Opin. Genet. Dev. 8: 185-193.
Was this article helpful?