Controling telomere replication - telomerase recruitment and the unwinding of telomeric G-quadruplex structure
Final Report Abstract
Telomeres are simple G-rich repetitive sequences at the ends of eukaryotic chromosomes and required for genome stability. To avoid chromosome shortening during replication of the linear chromosomal DNA telomerase adds telomeric repeats to the ends. It was shown first in ciliated protozoa that telomeres adopt the G-quadruplex structure and that this structure will inhibit the action of telomerase. Therefore G-quadruplex structure has to be resolved during replication and telomerase has to be recruited to the telomeres. Understanding the regulation of these two processes was the aim of the present project. During DNA fragmentation or programmed DNA elimination telomeres have to be added de novo. In the course of macronuclear differentiation in stichotrichous ciliates telomeres are added to about 16.000 DNA-molecules and therefore ciliates represent an ideal model system to study this process. Within this project we could identify a RecQ-like helicase which seems to be involved in resolving telomeric G-quadruplex structure. This helicase is associated with telomerase and recruited to replicating telomeres by this enzyme. There was some evidence that the telomere end binding protein TEBPβ is somehow involved in telomerase recruitment similar to its human homolog TPP1. TEBPβ becomes phosphorylated during S-phase and we no could demonstrate that only the phosphorylated protein recruits telomerase. We and the group of Daniela Rhodes spend considerable effort to express the full length telomerase but were not able to get sufficient material for experimental work. There is some evidence that the TEN domain of telomerase interacts with TPP1 in human cells. We therefore expressed this domain and could show by various techniques that it indeed interacts with phosphorylated TEBPβ. To characterize this interaction in more details the TEN domain was digested with V8 protease, incubated with the phosphorylated telomere complex and precipitated with an anti-TEBPβ antibody. The precipitate was probed with an anti-telomerase antibody and a 4.6 kD TEN fragment was identified in the precipitate. Interestingly this 4.6 kD fragment contains some residues shown to be important for telomerase processivity in human telomerase. These residues became mutated and expressed. All material was send to our co-operation partner Prof. Daniela Rhodes, Singapore where the structure of the telomere-TEN complex will be analyzed in detail. Excision of DNA-fragments during macronuclear differentiation in ciliates seems to be regulated by snRNA. However, for the precision of this process, for reordering DNA-fragments and for specific amplification of macronuclear nanochromosomes long macronuclear derived RNA templates seem to be required. Interestingly these template molecules carry telomere sequences at their ends leading to the hypothesis that they may also be involved in de novo telomere addition. This hypothesis could be verified by injecting template molecules carrying mutant telomeric sequences which later could be shown to be present in the vegetative cells. This is the first report how telomeres may be added de novo to fragmented chromosomes and might be a guideline for studies in other organisms. In summary, due to technical (and personnel) problems we could not solve all questions raised in our original proposal. However, the results received will now allow to study for the first time the structure of the telomere-telomerase complex which will be most important for basic but also medical science. Our surprising observation that long RNA is involved in de novo addition of telomeres may be relevant for experiments in other organisms, where programmed DNA elimination takes place during embryogenesis.
Publications
- (2012) A telomeraseassociated RecQ protein-like helicase resolves telomeric G-quadruplex structures during replication. Gene, 497(2), 147-54
Postberg, J., Tsytlonok, M., Sparvoli, D., Rhodes, D., Lipps, H.J.
(See online at https://doi.org/10.1016/j.gene.2012.01.068) - (2015) G-quadruplexes and their regulatory roles in biology. Nucl. Acids Res. 43, 8627-8637
Rhodes, D., Lipps, H.J.
(See online at https://doi.org/10.1093/nar/gkv862) - (2016) Guanine quadruplex structures localize to heterochromatin. Nucleic Acids Res. 2016 44. 152-63
Hoffmann, R.F., Moshkin, Y.M., Mouton, S., Grzeschik, N,A., Kalicharan, R.D., Kuipers, J., Wolters, A.H., Nishida, K., Romashchenko, A.V., Postberg, J., Lipps. H., Berezikov, E., Sibon, O.C., Giepmans, B.N., Lansdorp, P.M.
(See online at https://doi.org/10.1093/nar/gkv900) - (2016) RNA-template dependent de novo telomere addition. RNA Biology Jan. 19 [Epub ahead of print]
Fuhrmann, G., Jönsson , F., Weil, P. Postberg, J., Lipps, H.J.
(See online at https://doi.org/10.1080/15476286.2015.1134414) - (2016) Telomerase–recruitment und de novo Addition von Telomeren im stichotrichen Ciliaten Stylonychia lemnae. Dissertation, Fakultät für Gesundheit der Universität Witten-Herdecke
Fuhrmann, G.
