GLC7 is an essential gene in Saccharomyces cerevisiae that encodes the catalytic subunit of protein phosphatase-1. The Glc7 protein regulates diverse cellular processes including mitosis. We have identified a Glc7 pathway that controls cell division; however, the components of this pathway are not fully understood. Glc7 is conditionally activated by phospho-Glc8, which is phosphorylated by the cyclin dependent kinase, Pho85, associated with Pcl6 and Pcl7 (two of its ten cyclins). Our knowledge about these two cyclins is limited. Therefore, our goal was to determine the input(s) that regulate Pcl6 and Pcl7. This will provide us with valuable information about the conditional activation of Glc7 by phospho-Glc8.
We determined Pcl6 to be more stable (t ½ = 8.4 hr. ± 1.2) than Pcl7 (t ½ = 52 min ± 7). We confirmed and discovered that not only does Elongin C (Elc1) stabilize Pcl6, but it also stabilizes Pcl7 as well. A null mutation of elc1 compromises the in vivo function of Pcl6 and Pcl7 in cell growth and in DNA damage response to 4-nitroquinoline oxide (4-NQO). Since Elc1 is found in two complexes that function in nucleotide excision repair pathway (NEF4 and Ela1 containing complex), we hypothesized the Pcl6 and Pcl7 levels are induced by DNA damage and are controlled by the Elc1 containing complexes. We tested our hypothesis by studying the stability of the both cyclins in NER mutants and we discovered that the NEF4 and a non-Elc1 containing complex, NEF2, control the stability of both cyclins. Furthermore, the Ela1 containing complex does not have a significant effect on the stability of both cyclins since the deletion of ela1 did not compromise the in vivo function of Pcl6 and Pcl7.
The NEF4 functions as an E3 ubiquitin ligase and have been speculated to interact with Rad23, since its substrate Rad4, physically interact react with Rad23 and forms the NEF2 complex. We propose that, in the presence of DNA damage, the main effector kinase of the DNA damage response pathway, activates the NEF4 and NEF2 via phosphorylation and consequently stabilize Pcl6 and Pcl7 by targeting the Pcl6 and Pcl7 ubiquitin ligases for destruction. Hence both cyclins become stable to function in cell recovery from the DNA damage. Our data supports this hypothesis by revealing that DNA damage increases the levels of both cyclins. The level of Pcl6 is regulated post-translationally, unlike Pcl7, which is regulated transcriptionally upon DNA damage. We also show that both cyclins function in cell response to 4-NQO DNA damage and cell growth.
In summary, our data reveal that two of the NER complexes, NEF4 and NEF2, both regulate the stability of Pcl6 and Pcl7, indicating that DNA damage regulates both cyclins. Hence, DNA damage is an input that controls the conditional activation of Glc7 by phospho-Glc8.
|Advisor:||Cannon, John F.|
|School:||University of Missouri - Columbia|
|School Location:||United States -- Missouri|
|Source:||DAI-B 73/03, Dissertation Abstracts International|
|Keywords:||Glc7, Pcl6, Pcl7, Saccharomyces cerevisiae|
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