What is the role of Doa1 in cell cycle response to DNA damage in Saccharomyces cerevisiae?
Cells have created many pathways to protect themselves from DNA damage and have ways to detect and respond to this damage. Phosphorylation has been seen to be involved in the DNA damage response of Saccharomyces cerevisiae during its posttranslational
modification of proteins, but other modifications have come to light. Ubiquitination (Ub) is one of these posttranslational modifications which involves three enzymes (E1, E2, E3) that results in the binding of a target substrate and an ubiquitin molecule (Fig. 1). This molecule is what labels the protein as one that needs to be destroyed (Lis, E.T., Romesberg, F.E., 2006). The binding of ubiquitin allows for the labeled protein to be dissembled in the proteasome. The details of Ubiquitin signalling in general can be seen visually in the video below.
Researchers have found that Doa1 has 5 WD repeats that are in many proteins involved in transcription, cell cycle control, cell fate determination and RNA metabolism (Lis, E.T., Romesberg, F.E., 2006). Doa1 was found while screening for mutants that could not degrade model proteasome substrates. In yeast, lack of Doa1 has caused sensitivity to caffeine, growth at increased temperatures and other DNA damaging agents (Ren J, Pashkova N, Winistorfer S, Piper RC., 2008).
Doa1 is a protein that helps to control ubiquitin concentration and is required in protein degradation using ubiquitin. These repeats bind to proteins that contain UBX domains, which is ubiquin regulatory X. A major finding showed that LUB1, the Schizosaccharomyces pombe homologue of Doa1, is involved in the stress response of S.pombe by creating Ub for proteasome-mediated protein degradation (Lis, E.T., Romesberg, F.E., 2006). This gave a hint to researchers and pushed them to try to understand what role Doa1 plays.
Interaction of Doa1 and Ub
To determine the role of Doa1 in the S. cerevisiae DNA damage response, a yeast two-hybrid screen, used to establish protein-protein interactions, was performed against an S. cerevisiae genomic library (Fig. 2). After the library plasmids were sequenced, four proteins were seen to interact with Doa1: Ubi4, Nfi1, Ubx5 and Ubx7. In the presence of MMS, showing a damaged screen, Ub14 and Ubp7 proteins interacted with Doa1 (Lis, E.T., Romesberg, F.E., 2006). This confirmed that Doa1 binds to Ub.
Doa1 in H2B Ubiquitination
The role of Doa1 in H2B ubiquitination was also studied. It was seen that the deletion of UBP8 or UBP10, histone H2B deubiquitinases, decreases the sensitivity of doa1Δ cells to DNA damage. Also, deleting one of the genes encoding histone H2B, HTB2, has the same affect on doa1Δ cells. Without Doa1 present, H2B-Ub levels decreased and after MMS was added, doa1Δ cells had a H2B ubiquitination defect (Fig. 3D). Expression of Ub did not affect H2B ubiquitination defect in the mutant strain (Fig. 3E) (Lis, E.T., Romesberg, F.E., 2006). This implies that Doa1 plays a role in the ubiquitination of histone H2B. Doa1 helps to maintain H2B ubiquitination levels, which controls the transcriptional response during DNA damage.
Although more research should be done to further explain these results, from this experiment Doa1 can be said to provide Ub to help with DNA damage and is necessary in H2B modification. This study does an excellent job of introducing the topic in a well organized and detailed fashion using figures and images to support the point.
Interaction of Doa1 and Hse1
Doa1 was found while screening for mutants that could not degrade model proteasome substrates. In yeast, lack of Doa1 has caused sensitivity to caffeine, growth at increased temperatures and other DNA damaging agents.
The Vps27-Hse1 complex is the Ub sorting receptor on the surface of early endosomes and with the help of this receptor, Ub can direct membrane proteins to the luminal vesicles of multivesicular bodies (MVBs) (Ren J, Pashkova N, Winistorfer S, Piper RC., 2008). These multivesicular bodies fuse to lysosomes to transport the proteins to the lysosomal lumen for degredation. Hse1 has been seen to interact with Doa1 to help sort these proteins into MVBs. The binding of Hse1 to Doa1 was assessed and it was found that Doa1 does bind specifically to the SH3 domain of Hse1 (Fig. 4A).
What Happens When Doa1 is Lacking?
Two MVB markers were used, Cps1 with green florescent protein and Cps1 fused to ubiquin and green florescent protein, to see the effects of the loss of Doa1. Cpd1 is a vacuolar protease that uses the MVB pathway. In the experiment, mutant doa1 could not sort GFP-Cps1 into the vacuole lumen and the GFP-Cps1 collected instead on the membrane of the vacuole (Ren J, Pashkova N, Winistorfer S, Piper RC., 2008). Ub-GFP-Cps1 on the other hand, could enter the vacuolar lumen in mutant doa1 cells because these proteins did not need ubiquitination for MVB sorting (Fig. 4C). These results showed that Doa1 is involved in MVB sorting by helping proteins to undergo ubiquitination to enter the degradation process.
Importance of Knowing the Role of Doa1
Why do we need to know how Doa1 affects pathways in S. cerevisiae? With many factors being introduced to the yeast cell, there are several ways these factors can cause harm. When harm has occurred to the cell causing DNA damage, the cell has to have a way to ‘cleanup’ the damage. Knowing what Doa1 does and how it performs these actions in the cell and how it contributes to this clean up effort and further our knowledge on the Ubiquitination pathway. One can also take a step ahead and focus on the Ubiquitination pathway in other organisms such as humans. Researchers have started studying the role of this pathway in Parkinson’s diesease and in other neurodegenerative disorders (Pickart, C. M., Ross, C. A., 2004)
Recent studies have shown that with Doa1 missing in yeast cells, the ubiquitination pathway cannot be completed. The binding of Doa1 to Ub is possible through the Cdc48 adaptor and not only does this help in the ubiquitination pathway, but it also aids in histone ubquitination for the transcriptional response during DNA damage (Mullally, J.E., Chernova, T., Wilkinson, K.D., 2006). With the help of GFP-Cps1, researchers determined the need for Doa1 in the MVB pathway to bring membrane proteins to lysosomes for degradation. These studies furthered the belief that Doa1 plays a major role in the degradation of proteins in both the proteasome and lysosome with evidence supporting this belief.
Further experiments can be done to understand how ubiquitination is regulated in S. cerevisiae and how it is used after DNA damage. What other proteins are inv0lved in this process and which ones work with Doa1? What happens when modification of these proteins are present in yeast and mammalian cells? Are these proteins seen in human cells and will studies be conducted to further the knowledge of the degradation pathway? These questions and questions like these could be answered from studies conducted along the lines of the two studies presented above.
The studies mentioned above did not present any weaknesses that could be seen. The research was sound and the evidence provided support to the authors arguments.
- Doa1 is necessary for protein degradation in budding yeast.
- Doa1 binds to the ubiquitin molecule to undergo the ubiquitin pathway.
- Ubiquitination is affected in histone H2B molecules when Doa1 is missing as ubiquitination levels are significantly decreased.
- Doa1 binds to Hse1 to transport membrane proteins to the lysosome for degredation.
- When Doa1 is lacking, ubiquitination is not present and this causes proteins to sit outside of the lumen and not degrade in the lysosome.
- The study of Doa1 can help researchers map out the ubiquitination pathway in humans and see how modification of this pathway can help find cures of many known diseases.
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Online Resource Links
Lis, E.T., Romesberg, F.E. (2006). Role of Doa1 in the Saccharomyces cerevisiae DNA Damage Response. Mol. Cell. Biol.,I 26(11), 4122–4133.
Mullally, J.E., Chernova, T., Wilkinson, K.D. (2006). Doa1 is a Cdc48 adapter that possesses a novel ubiquitin binding domain. Mol. Cell. Biol., 26(3), 822-830.
Pickart, C. M., Ross, C. A. (2004). The ubiquitin-proteasome pathway in Parkinson’s disease and other neurodegenerative diseases. Trends in Cell Biology, 14(12), 703-711.
Ren J, Pashkova N, Winistorfer S, Piper RC. (2008). DOA1/UFD3 Plays a Role in Sorting Ubiquitinated Membrane Proteins into Multivesicular Bodies. Journal of Biological Chemisty, 283(31), 21599-611.