PMC:1072800 / 14182-17131 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/1072800","sourcedb":"PMC","sourceid":"1072800","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1072800","text":"hOGG1 is sorted to the nucleoli during S-phase\nStudies of transiently transfected HeLa cells with the reporter protein EGFP fused to the C-terminal of hOGG1 have previously shown that the enzyme is transported to the nucleus (14). These studies revealed a heterogeneous population of cells displaying either homogeneous or reminiscent nucleolar localization of hOGG1. To confirm the nucleoli localization of EGFP-hOGG1, cells were fixed and nucleoli were visualized by confocal microscopy using antibodies against nucleolin. When the signals from EGFP-hOGG1 and anti-nucleolin were merged, EGFP-hOGG1 was found to be concentrated predominantly within the nucleoli as shown in Figure 2A. Furthermore, we established stable HeLa cell lines carrying either EGFP alone or EGFP-hOGG1 fusion proteins to further study the nuclear sublocalization of hOGG1 during the cell cycle. Overexpression of EGFP and EGFP-hOGG1 in the stable cell lines was examined in nuclear extracts by western blot using anti-OGG1 and anti-GFP specific antibodies. EGFP (30 kDa), EGFP-hOGG1, EGFP-hOGG1-Cys326 and EGFP-hOGG1-Glu326 (68 kDa) were recognized by the anti-GFP specific antibody (Figure 3A) while only EGFP-hOGG1, EGFP-hOGG1-Cys326 and EGFP-hOGG1-Glu326 (68 kDa) were recognized by the anti-OGG1 specific antibody (Figure 3B). Nuclear extracts from HeLa-EGFP and HeLa-EGFP-hOGG1 were tested for faPy DNA glycosylase activity to demonstrate the functionality of the fusion protein product. Results showed that hOGG1 fused to the EGFP was catalytically active (Figure 3C).\nCells were synchronized by nocodazole, which causes the cells to arrest at the G2/M boundary, or by the double-thymidine block, which arrest the cells at G1/S boundary. Cell samples were taken at different time points after release and cell-cycle progression was followed by flow cytometry. Samples were fixed and examined under a fluorescence microscope. Immediately after the removal of nocodazole most of the cells are in the G2 phase. At this time, EGFP-hOGG1 is mainly distributed evenly throughout the nucleus (Figure 4). Within the next 2 h, cells enter the M-phase, the nuclear envelope disassembles and EGFP-hOGG1 associates with chromatin (Figure 4). At 2–8 h after nocodazole removal (data not shown), and at the time of double-thymidine block release, the cells are in G1, and EGFP-hOGG1 is mainly homogeneously distributed to the nucleus (Figure 4). However, as the cells progress through the S-phase, 4 h after double-thymidine block release, in most of the cells, EGFP-hOGG1 accumulates within the nucleoli (Figure 4). In addition, to confirm the localization of EGFP-hOGG1 to the nucleoli during S-phase, we visualized sites of replication using anti-BrdU antibodies. As shown in Figure 2B, EGFP-hOGG1 is associated with the nucleoli during S-phase. Control cells carrying only EGFP stained green both in the cytoplasm and in the nucleus throughout the cell cycle (data not shown).","divisions":[{"label":"Title","span":{"begin":0,"end":46}}],"tracks":[{"project":"2_test","denotations":[{"id":"15800211-9321410-76712980","span":{"begin":226,"end":228},"obj":"9321410"}],"attributes":[{"subj":"15800211-9321410-76712980","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#da93ec","default":true}]}]}}