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Chromosoma

, Volume 122, Issue 1–2, pp 55–66 | Cite as

Drosophila SUUR protein associates with PCNA and binds chromatin in a cell cycle-dependent manner

  • Tatyana D. Kolesnikova
  • Olga V. Posukh
  • Eugeniya N. Andreyeva
  • Darya S. Bebyakina
  • Anton V. Ivankin
  • Igor F. Zhimulev
Research Article

Abstract

Drosophila SUUR (Suppressor of UnderReplication) protein was shown to regulate the DNA replication elongation process in endocycling cells. This protein is also known to be the component of silent chromatin in polyploid and diploid cells. To mark the different cell cycle stages, we used immunostaining patterns of PCNA, the main structural component of replication fork. We demonstrate that SUUR chromatin binding is dynamic throughout the endocyle in Drosophila salivary glands. We observed that SUUR chromosomal localization changed along with PCNA pattern and these proteins largely co-localized during the late S-phase in salivary glands. The hypothesized interaction between SUUR and PCNA was confirmed by co-immunoprecipitation from embryonic nuclear extracts. Our findings support the idea that the effect of SUUR on replication elongation depends on the cell cycle stage and can be mediated through its physical interaction with replication fork.

Keywords

Salivary Gland Replication Fork Polytene Chromosome Replication Machinery Cell Cycle Stage 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We thank S. Kulemzin for help with the FPLC assay. We are grateful to Prof. H. Saumweber for providing anti-tubulin antibodies, S. Elgin for providing anti-HP1 antibodies, and to A.A. Alekseyenko for providing MSL3-TAP flies. We thank E. Belyaeva for critical reading and comments on the manuscript. The research was funded by Program of RAS “Molecular and Cellular Biology” 22.4; Integration project of SB RAS #82, and the Russian Foundation for Basic Research (#12-04-31777-mol_a and #12-04-00874-а).

Supplementary material

412_2012_390_MOESM1_ESM.jpg (254 kb)
Fig. S1 PCNA co-localizes with BrdU pattern after 30 min pulse incorporation Salivary gland polytene chromosomes immunostained for BrdU (red) and PCNA (green). Arrows indicate nuclei at early, middle, and late S-phase stages.
412_2012_390_MOESM2_ESM.jpg (717 kb)
Fig. S2 SUUR and PCNA co-localize during late S-phase Proximal part of 2R chromosome immunostained for SUUR (green) and PCNA (red) at different endocycle stages: a - early S-phase, PCNA labels all chromosomal regions except the thick bands, no SUUR signal is detected; b – early-to-late S-phase transition, both PCNA and SUUR signals are present in most chromosomal bands; c-d - late S-phase stages, SUUR and PCNA patterns fully co-localize; e - G-phase, PCNA signal is not detected and SUUR is present only in chromocenter. The Roman figures indicate S-phase stages as described in Fig. 1. Here we divided the stage III into early (III-1) and late (III-2).
412_2012_390_MOESM3_ESM.jpg (557 kb)
Fig. S3 E45 anti-SUUR antibodies display no signals in chromosomes during endocycle SUUR (green) and PCNA (red) double-staining of salivary gland polytene chromosomes. PCNA patterns mark different endocycle stages (I-V) according to Fig. 1.
412_2012_390_MOESM4_ESM.jpg (787 kb)
Fig. S4 SUUR is detected in numerous sites on chromosomal arms upon HU treatment Salivary gland cells arrest in S-phase after HU treatment. a - PCNA immunostaining (red) confirms that HU treatment leads to the accumulation of the nuclei at early S-phase stages (arrows). b - Early S-phase nuclei (arrows) display multiple SUUR signals (green) on chromosomal arms that are less intense than chromocenter staining; late S-phase nucleus (arrowhead) has fewer SUUR signals, however, their intensity is comparable to chromocenter staining, as seen in the nuclei without HU treatment. c - Close-up of the marked region in (a,b): incomplete co-localization of SUUR and PCNA in early S-phase chromosomes.
412_2012_390_MOESM5_ESM.jpg (380 kb)
Fig. S5 Dynamic chromosomal localization of SUUR and PCNA during endocycle in 4x salivary glands Nuclei of the same salivary gland at different endocycle stages stained for PCNA and SUUR, photographed at the same exposure: a-e - S-phase stages from early to late; f - G-phase. Increased SUUR concentrations further emphasize correlation with PCNA (b-d). The Roman figures indicate S-phase stages as described in Fig. 1. Here we subdivided the stage III into early (III-1) and late (III-2).
412_2012_390_MOESM6_ESM.jpg (682 kb)
Fig. S6 SUUR chromosomal binding patterns during late S-phase in 4x genotype Late replication sites and SUUR localization were previously mapped on wild-type polytene chromosomes (Zhimulev et al., 2003a). Authors considered nuclei to be late replicating, if 3R chromosome displayed 40-45 [3H]thymidine incorporation sites. Using this approach, the fragment of late S-phase X chromosome between 1A-11A regions was shown to have 26 late replication sites. SUUR localized to only 16 of those sites (1AB, 3C, 4D, 4D, 4E, 5DE, 6A, 7B, 7C, 7E, 8B, 8E, 9A, 10A, 10B, 11A6-9). Six sites (3С3-5, 3D1-2, 5D1-6, 7B1-2, 10B1-2, 11A6-9) had chromosomal breaks due to under-replication, indicating that replication of those regions is the latest. It was also shown that in 4xSuUR + genotype, SUUR localizes to all late replication sites. Here we demonstrate chromosomal localization of PCNA and SUUR at different S-phase stages along the 1A-11A fragment of X chromosome in the nuclei of the same salivary gland of 4xSuUR + genotype: a - Beginning of late S-phase: PCNA binds 30 chromosomal sites and co-localizes with SUUR that binds 27 sites corresponding to the late replication regions (Zhimulev et al., 2003a). b - Late S-phase: PCNA and SUUR fully co-localize in 13 chromosomal sites that correspond to wild-type SUUR binding sites (Zhimulev et al., 2003a). c - The latest S-phase: SUUR is detected in several chromosomal regions, whereas PCNA is only detected in 3C and 11A, the regions corresponding to the latest replication sites on X chromosome. Thus, we show that SUUR pattern changes during endocycle in 4xSuUR + genotype similar to the wild-type. SUUR co-localizes with PCNA during late S-phase. The number of SUUR binding sites decreases with the progression of the S-phase (chromosomal bands replicate in a distinct order).
412_2012_390_MOESM7_ESM.jpg (510 kb)
Fig. S7 HP1 binding pattern on polytene chromosomes does not significantly change throughout endocycle HP1 (green) and PCNA (red) double-staining of salivary gland polytene chromosomes. PCNA patterns mark different endocycle stages (I-V) according to Fig. 1.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Tatyana D. Kolesnikova
    • 1
  • Olga V. Posukh
    • 1
  • Eugeniya N. Andreyeva
    • 1
  • Darya S. Bebyakina
    • 1
  • Anton V. Ivankin
    • 1
  • Igor F. Zhimulev
    • 1
  1. 1.Institute of Molecular and Cellular BiologySiberian Branch of Russian Academy of SciencesNovosibirskRussia

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