Ultrastructural investigation of DNA replication in Drosophila polytene chromosomes via super-resolution microscopy
General Genetics and Functional Genome Biology
Structural Biology
Final Report Abstract
In the project we aimed to visualize the ultrastructure and dynamics of DNA replication in Drosophila polytene chromosomes by applying spatial super-resolution structured illumination microscopy (3D-SIM). We explored the spatio-temporal organization of replication in distinct individual chromosome regions. Methods have been optimized to image replication after EdU incorporation and immunolabeling with antibodies against the replication fork protein PCNA. Based on a Drosophila line carrying the hsp70-CycE transgene an approach was developed to induce the S phase in salivary gland cells within not more than 20 minutes. Thus, we were able to analyze the dynamics of replication initiation during the early S phase. The hypothesis formulated earlier by Kolesnikova et al. (2018), that the initiation of replication in polytene chromosomes occurs in intervals, was confirmed, at which it was found to be probabilistic. In addition, we found regions where replication initiation occurs very efficiently in the first minutes of the S phase on most strands of the polytene chromosome and regions where it does not occur simultaneously, with a time shift of tens of minutes. We localized about 40 very efficient early replication initiation sites. Most of these sites coincided with the loci of ecdysone-dependent genes. We developed new approaches for visualizing transcriptionally active genes by 3D-SIM. The simultaneous detection of RNA polymerase II phosphorylated at serine 5 and 2 indicating transcriptional initiation and elongation, respectively, allows to visualize long genes during their transcriptional progression. Thus, we also visualized the direction of transcription, especially for genes located in puffs. In addition to classical stripe SIM, we applied Lattice SIM² realized by the improved Elyra 7 microscope system resolving up to 60 nm. With it, we detected the double amount of RNA polymerase II molecules in transcriptional active puffs. The distribution of several proteins and histone modifications was visualized in polytene chromosomes by 3D-SIM. The effect of mutations in the Rif1 gene on morphology, underreplication, and replication timing in the pericentromeric heterochromatin of polytene chromosomes of D. melanogaster was studied. Via 3D-SIM, we examined the unique organization of the nucleolus, which is fully polytenized in Rif1 mutants. Protocols and software have been developed to analyze replication in spatially preserved nuclei of salivary glands. For it, we applied 3D-SIM to detect replication marks on semi-thin sections, as well as the sequential study of ultra-thin sections via light and electron microscopy (correlation microscopy).
Publications
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Ultrastructural investigation of early replication in Drosophila polytene chromosomes. Biopolymers and Cell, 35(3), 187-188.
Kolesnikova, T. D.; Pokholkova, G. V.; Kolodyazhnaya, A. V. & Schubert, V.
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Effects of Mutations in the Drosophila melanogaster Rif1 Gene on the Replication and Underreplication of Pericentromeric Heterochromatin in Salivary Gland Polytene Chromosomes. Cells, 9(6), 1501.
Kolesnikova, Tatyana D.; Kolodyazhnaya, Alexandra V.; Pokholkova, Galina V.; Schubert, Veit; Dovgan, Viktoria V.; Romanenko, Svetlana A.; Prokopov, Dmitry Yu. & Zhimulev, Igor F.
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Super-resolution microscopy reveals the early replication dynamics in Drosophila polytene chromosomes. International Conference “Drosophila in genetics and medicine”, St Petersburg – Gatchina, 30 September 2020
Kolesnikova T.D., Pokholkova G.V., Kolodyazhnaya A.V., Dovgan V.V., Schubert V. & Zhimulev I.F.
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Structured illumination microscopy reveals the replication initiation dynamics in Drosophila polytene chromosomes. 62nd Annual Drosophila Research Conference, 23 March – 1 April 2021
Kolesnikova T.D., Schubert V., Pokholkova G.V. & Dovgan V.V.
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A Spontaneous Inversion of the X Chromosome Heterochromatin Provides a Tool for Studying the Structure and Activity of the Nucleolus in Drosophila melanogaster. Cells, 11(23), 3872.
Kolesnikova, Tatyana D.; Klenov, Mikhail S.; Nokhova, Alina R.; Lavrov, Sergey A.; Pokholkova, Galina V.; Schubert, Veit; Maltseva, Svetlana V.; Cook, Kevin R.; Dixon, Michael J. & Zhimulev, Igor F.
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Super-resolution microscopy reveals stochastic initiation of replication in Drosophila polytene chromosomes. Chromosome Research, 30(4), 361-383.
Kolesnikova, Tatyana D.; Pokholkova, Galina V.; Dovgan, Viktoria V.; Zhimulev, Igor F. & Schubert, Veit
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Взаимосвязь между особенностями инициации репликации транскрипционной активностью в политенных хромосомах Drosophila melanogaster. III Объединенный научный форум физиологов, биохимиков и молекулярных биологов;Научные труды том 2, Сочи – Дагомыс, Russia 3–7 October 2022
Kolesnikova T.D., Dovgan V.V., Pokholkova G.V. & Schubert V.
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Clusters of ecdysonedependent enhancers contain highly efficient early replication initiation sites in Drosophila polytene chromosomes. Cold Spring Harbor Conf. Sept 2023
Kolesnikova T.D., Schubert V., Dovgan V.V. & Vorobyeva N.E.
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Drosophila polytene chromosomes as a tool for studying the probabilistic nature of replication initiation. Conference Chromosome 2023, Novosibirsk, Russia
Kolesnikova T.D., Vorobyeva N.E., Dovgan V.V. & Schubert V.
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Сайты очень эффективной ранней инициации репликации в политенных хромосомах слюнных желез Drosophila melanogaster связаны с кластерами экдизонзависимых энхансеров. Kонференция с международным участием „Дрозофила 2023“, St Petersburg – Gatchina, 3 – 6 October 2023
Kolesnikova T.D., Vorobyeva N.E., Dovgan V.V., Pokholkova G.V. & Schubert V.
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Global nuclear reorganization during heterochromatin replication in the giant‐genome plant Nigella damascena L.. The Plant Journal, 120(4), 1508-1521.
Arifulin, Eugene A.; Sorokin, Dmitry V.; Anoshina, Nadezhda A.; Kuznetsova, Maria A.; Valyaeva, Anna A.; Potashnikova, Daria M.; Omelchenko, Denis O.; Schubert, Veit; Kolesnikova, Tatyana D. & Sheval, Eugene V.
