Posttranslational attachment of the ubiquitin-related protein SUMO to other proteins participates in many vital cellular processes including transcription, signaling and maintenance of genome integrity. However, the mechanisms by which SUMO affects its substrates are poorly understood. This project will determine the role(s) of SUMO in controlling the copy number of the 2 micron circle, a selfish DNA element that resides in budding yeast. SUMO pathway mutants accumulate such high copy numbers of 2 micron that it causes cell death. Preliminary data suggest that this effect is caused by an aberrant activity of Flp recombinase, a protein encoded by 2 micron. This results, at least partly, from deficient SUMO attachment to Flp itself. Importantly, Flp sumoylation requires not only SUMO pathway enzymes, but also other features of 2 micron. This suggests that Flp may become sumoylated as a part of its functional cycle at the DNA.
This hypothesis will be tested by a) defining the role of DNA binding in Flp sumoylation and b) determining whether sumoylation targets a specfic subpopulation of Flp for ubiquitin-dependent proteolysis via a SUMO-specific ubiquitin E3 ligase. This research will help solve a basic question in the SUMO field: how can SUMO affect protein function when most substrates are sumoylated at extremely low levels. Interdependency between enzyme activity and sumoylation would explain this observation, as would sumoylation-dependent proteolysis. A second set of experiments will test whether Flp causes abnormal levels of DNA damage in SUMO pathway mutants. The results of these experiments will shed light on the complex role of the SUMO pathway in maintaining genome stability.
One way that biological processes are regulated is via attachment of certain small proteins, including a protein called SUMO, to other proteins. SUMO is essential for cell growth and is involved in almost every major nuclear process, but the means by which SUMO alters the activity of the proteins to which it is attached is not clear. This project will examine how SUMO attachment controls the activity of the Flp protein and should provide insight into how SUMO acts in other crucial processes as well. The investigator has received a Dean's Citation for her commitment to education and through this award she will continue her contributions in that area. Part of this project will be conducted by a graduate student as part of her Ph.D. thesis research, and undergraduate students will participate in this project through a summer research program. Students and postdoctoral researchers performing this work will present their findings at departmental, city-wide, and international meetings and through scientific publications.
The goal of this project was to understand how budding yeast use a small modifier protein called SUMO?to control levels of a parasitic DNA element called the 2 micron circle. ?SUMO is a highly conserved protein that is found in all eukaryotes, including humans. SUMO acts through its ability to become attached to other proteins. This allows it to regulate the activities of these other proteins. ?The importance of SUMO is demonstrated by the fact that it becomes attached to many different proteins and is required for cell viability. However, there are still many questions that remain to be answered about how SUMO attachment is regulated and about the functional roles of SUMO in cellular activities. ?Many studies have suggested that a major set of SUMO functions are related to maintaining the stability of the genome. ?Our studies have investigated the role of SUMO in controlling stability of the 2 micron circle, which serves as a useful model for functions of SUMO in stability of the entire genome.? The first finding from this work was that SUMO attachment to the protein Flp, which is encoded by 2 micron and is required for control of 2 micron copy number, prevents accumulation of an aberrant form of 2 micron that promotes uncontrolled copying of this DNA element. ?The Flp protein acts through an enzyme mechanism that is related to that of some normal cellular enzymes, which may suggest that the activity of these proteins may also be influenced by SUMO attachment. ?SUMO attachment to the Flp protein targets Flp ?for destruction, which is likely an important aspect of SUMO function in this pathway. While SUMO attachment to Flp is important, we also found that preventing SUMO attachment to the Flp protein alone did not have the same effect on 2 micron behavior as did reducing total SUMO attachment. ?This suggested that SUMO has additional roles in 2 micron control other than modifying Flp. One candidate for this other role of SUMO would be an activity related to DNA replication or repair. ?The aberrant behavior of 2 micron that is seen when SUMO attachment is deficient involves damage to, and repair of, the 2 micron DNA.? Our next finding was that SUMO attachment participates in a DNA repair pathway called nucleotide excision repair (NER), which removes bulky DNA lesions such as those caused by the UV rays in sunlight. ?This work showed that several different nucleotide excision repair proteins become attached to SUMO when cells are exposed to UV light. ?It also showed that when downstream steps of the repair pathway are blocked, SUMO-modified forms of upstream factors accumulate. ?This result suggests that SUMO attachment to individual factors takes place as they carry out their function in the repair pathway. ?Preventing SUMO attachment to one heavily SUMO-modified NER protein did not affect the rate of UV-damage repair. ?This would be consistent with a model where SUMO can function through attachment to any one of several NER proteins, and does not necessarily have to be attached to any one individual factor. Finally, we observed during this work that SUMO attachment is extremely sensitive to the nutritional state of the cell, such that ~80% of SUMO conjugates disappear within minutes when the cell is starved. ?Our work showed that this effect is a direct effect of a low cellular energy charge on the SUMO attachment mechanism, not a secondary effect of a nutritional signaling pathway. ?This results suggests that some effects of conditions such as glucose starvation or oxygen deprivation might be caused by reduced SUMO attachment under these circumstances.? This project served as a training opportunity for one Ph.D student, two undergraduates who worked on it for multiple summers, and for three new college graduates who worked as laboratory technicians. ?This work provided training in genetics, molecular biology and biochemistry, as well as in developing and testing scientific hypotheses. Participation in this project has advanced the scientific/professional careers of all of these beginning scientists. The Ph.D student is now working as a postdoc in another lab with the goal of a teaching career, and one of the undergraduates has now graduated, and is doing research in another group here at Jefferson. ?Two of the technicians have gone on to medical school. ?The other technician is currently working on her Master's project in my lab. Work from this project has been presented in seminars and at national and international meetings by both the PI and the Ph.D. student. ?Strains and plasmids generated as a result of this project have been distributed to laboratories world-wide.
