Untersuchungen zum Einfluss von telomerassoziierten Proteinkomplexen auf die Regulation und Struktur der VSG Expression Site in Trypanosoma brucei
Zusammenfassung der Projektergebnisse
In summary, we systematically depleted all 21 candidates identified in the previous project by RNAi and evaluated their influence on VSG regulation by proteome profiling and MS analysis of VSG proteins. We anticipated to find a cohort of proteins that are involved in antigenic variation and planned to evaluate their exact function, their interaction with other known telomere-associated proteins and putative roles in telomere maintenance. To our surprise, we could only identify one protein, PollE, which is involved in VSG regulation and genome maintenance. In collaboration with the McCulloch group, we analysed PollE in details including high-resolution imaging, MS analysis, DNA damage and cell viability assays and evaluated its role in chromosome segregation, DNA repair, genome maintenance and VSG regulation. Additionally, we evaluated the protein composition of the telomere complex by intensive interactomic studies with tagged candidates and in vitro assays. We have confirmed previously known interactions of these proteins by reciprocal affinity purification and were able to improve the knowledge about their interaction pattern. This leads to the suggestion that the two translesion polymerases PPL2 and PollE are strongly connected while the TbTRF-TbTIF2- TbRAP1 complex interacts closely with TelAP1, TelAP2 and TelAP3. Through reciprocal co lmmunoprecipitations of TelAP1 and TbTRF in TelAP2 depleted cells, we validated the interaction of TelAP1 and TelAP2 and could show that TelAP2 tethers TelAP1 to the rest of the telomeric protein complex. We did not apply for a continuation of this project because we believe our study was based on our available techniques comprehensive and we do not anticipate further candidates involved in antigenic variation within this group of proteins.
Projektbezogene Publikationen (Auswahl)
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Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation. Nucleic Acids Research, 48(17), 9660-9680.
Leal, Andrea Zurita; Schwebs, Marie; Briggs, Emma; Weisert, Nadine; Reis, Helena; Lemgruber, Leandro; Luko, Katarina; Wilkes, Jonathan; Butter, Falk; McCulloch, Richard & Janzen, Christian J.
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Quantification of RNA Polymerase I transcriptional attenuation at the active VSG expression site in Trypanosoma brucei. Cold Spring Harbor Laboratory.
Weisert, Nadine; Thein, Klara; Reis, Helena & Janzen, Christian J.
