Chromosome Segregation Defects and Genome Instability Occur in Cells with TOP3A Mutations
Impaired dHJ decatenation can result in persistently entangled sister chromatids that impede chromosome segregation at mitosis.19 Therefore, we performed detailed characterization of the mitotic consequences of TopIIIα deficiency in primary fibroblasts from P1. DAPI staining demonstrated elevated amounts of chromatin bridges (control [C] = 0, parent = 0.3 ± 0.4, and P1 = 4 ± 0.6) and lagging chromatin and chromosomes (C = 0.9 ± 0.2, parent = 4 ± 0.7, and P1 = 9.2 ± 0.9) (Figures 4A and 4B), consistent with persisting chromosome entanglements. Additionally, increased numbers of ultrafine DNA bridges (UFBs) were revealed by immunostaining for PICH on DAPI-negative regions19 (C = 2.7 ± 0.3, parent = 13.1 ± 1.1, and P1 = 26.7 ± 3.8).
Figure 4 DNA Catenanes Persist into Mitosis in Cells with TOP3A Mutations, Leading to Chromosome Segregation Defects and Genome Instability
(A and B) Chromosome segregation is impaired in TOP3A-deficient primary fibroblasts. (A) Representative images of chromatin bridges, lagging DNA (DAPI), and UFBs (detected by the presence of PICH and absence of DAPI stain). (B) Quantification of chromatin bridges, lagging DNA, and PICH-positive UFBs scored in control, parental, and P1 mitotic fibroblasts staged at anaphase B (experiments ≥ 3, n > 50 cells, error bars = SEM). To enrich for mitotic cells, we treated fibroblasts with R03306 for 6 hr and released and fixed them after 45 min.
(C) TOP3A P1 fibroblasts display significantly elevated amounts of micronuclei. Top: representative picture of control and P1 fibroblasts. Bottom: quantification of micronucleus containing interphase cells (experiments ≥ 3, n > 500, error bars = SEM). To enrich for G1 cells, we released RO3306-treated cells into fresh media for 30 min and collected, re-seeded, and fixed prometaphase cells after 4–6 hr.
(D) P1 fibroblasts with TOP3A mutations display significantly elevated numbers of 53BP1 bodies in G1 nuclei. Top: representative images of 53BP1 foci (red) and DNA (DAPI). Bottom: quantification of cells with at least four 53BP1 foci in G1 nuclei (negative for cyclin A) (experiments ≥ 3, n > 500, error bars = SEM). Scale bar: 2 microns. Two-tailed t test was performed against parent cells.
We then examined the post-mitotic consequences of the observed chromosome segregation errors by enriching for G1 cells (Figures 4C and 4D). Micronuclei often arise from chromosome segregation errors,35 and analysis of DAPI-stained cells demonstrated substantially more micronucleated cells in the P1 fibroblast cell line than in control and parental cell lines (C = 0.4 ± 0.2, parent = 1.8 ± 0.5, and P1 = 7.5 ± 1.4). Elevated amounts of 53BP1 nuclear bodies were also seen in cyclin-A-negative (G1) cells, indicating the transmission of DNA damage from one cell cycle to the next36 (C = 0.8 ± 0.3, parent = 4.5 ± 0.5, and P1 = 11 ± 1.7). Altogether, we conclude that impaired dHJ decatenation in TopIIIα-deficient cells results in both abnormal recombination and mitotic errors that lead to accumulation of DNA damage. Because chromatin bridges and UFBs are also present in individuals affected by Bloom syndrome,19 these findings are concordant with a shared disease mechanism.