Smc5 depletion impedes cell cycle progression, induces DNA damage, and causes genomic instability in mouse embryonic fibroblasts

Smc5/6;mitosis;DNA damage;Biochemistry

Gaddipati, Himaja

Johns Hopkins University

关键词: Smc5/6;    mitosis;    DNA damage;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/44602/GADDIPATI-THESIS-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Introduction: The structural maintenance of chromosomes 5/6 (Smc5/6) complex plays a critical role in maintaining genomic integrity. More specifically, Smc5/6 is involved in DNA replication, DNA damage repair via homologous recombination (HR), and chromosome segregation. Although its function has been extensively studied in yeast, few studies have evaluated Smc5/6 in mammalian models. Based on existing literature, we hypothesized that Smc5/6-deficient mouse embryonic fibroblasts (MEF) would accumulate DNA damage and, as a result, demonstrate abnormal mitotic progression and show evidence of replication stress.Methods: We used transgenic mice harboring a floxed exon 4 of the Smc5 gene (Smc5flox/flox and Smc5+/del, Ert2-Cretg/0 ) to breed and establish immortalized MEF cell lines with genotypes of Smc5flox/del, Ert2-Cretg/0 (experimental), Smc5+/flox, Ert2-Cretg/0 (control #1), and Smc5flox/del (control #2). Smc5 exon 4 was deleted by addition of 0.2µM 4-OH tamoxifen for nine days. Deletion was confirmed by PCR and protein depletion by western blot. Cells were analyzed on day 3, 6 and 9.MEF growth characteristics and cell cycle progression were evaluated by performing cell counts and FACS analysis, respectively. We also used immunofluorescence microscopy to observe micronuclei formation and DNA bridges. Additionally, we analyzed Rad51, Sumo1, and Sumo2/3 after treating cells with hydroxyurea. Finally, we used western blot analysis to evaluate expression of the stress response marker, p53. Results: Smc5-depleted MEFs demonstrated several mitotic abnormalities. After six days of 4-OH tamoxifen treatment, we observed a sustained, two-fold decrease in cell proliferation compared to controls. FACS analysis showed delayed entry into S phase. DAPI staining of Smc5-depleted cells showed 12% increase in micronuclei formation and 33% increase in DNA bridges. Hydroxyurea treated cells showed an accumulation of Rad51 foci, suggesting impaired HR mechanisms. Mutation of Smc5 also resulted in a decline in Sumo1 but not Sumo2/3 foci. Lastly, western blot analysis showed significant p53 upregulation.Conclusions:For the first time, we have demonstrated the importance of the Smc5/6 complex in somatic mouse cells. Smc5 depletion in MEF cells compromises genomic integrity, affects cell cycle progression and leads to chromosome missegregation. We also demonstrate hypersensitivity to DNA damage agents and activation of the p53 pathway.