Topo IIIalpha and BLM act within the Fanconi anemia pathway in response to DNA-crosslinking agents

Abstract

The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of chromosome radial formation.

Fig. 1

Depletions in normal transformed human fibroblasts (GM639) and BS cells (GM08505). A Immunoblot of BLM, BRCA1 and FANCA depletions. Cells were transfected with siRNA, and harvested at the indicated times. β-Tubulin is shown to indicate loading. Cell lines are indicated under the blots. B Depletion of Topo IIα and Topo IIIα in normal (GM639), BS (GM08505) and FANCD2 (PD20) cells determined by qRT-PCR. Relative expression is in comparison to the non-depleted cell line.

Fig. 2

SCE formation in normal (A) and BS (B) cells. Normal (GM639) or BS cells (PSNG13 and GM08505) were treated with MMC as described in Materials and Methods and analyzed for SCE formation, with the indicated depletions. SCE are given per chromosome. + BLM indicates a complementation Bloom line, as described in Materials and Methods. The control data sets are from mock-transfected cells. Error bars represent the standard error of the mean.

Fig. 3

Chromosomal radial formation. A BS (GM08505) or B normal cells (GM639), were analyzed for radial formation as described in Materials and Methods with MMC (40 ng/ml) and the depletions indicated; the control data are from mock-transfected cells. Percent radials is the percent of cells with one or more radials. Error bars represent standard errors of the mean. No radials were observed in untreated normal cells.

Fig. 4

Survival of normal (GM639) cells depleted for BLM or Topo IIIα after treatment with varying amounts of MMC. Survival was determined by colony formation, normalized to a mock-treated control plate. Error bars represent the standard error of the mean.

Fig. 5

Cell cycle analysis with BLM or Topo IIIα depletion. Cells with or without MMC (80 ng/ml) were analyzed for DNA content. Cells are GM639 (normal) with control siRNA (A, B), GM08505 (BS) (C, D), GM639 with Topo IIIα siRNA (E, F), and GM6914 (FANCA) (G, H).

Fig. 6

Radial formation in FA cells depleted for BLM or Topo IIIα. FANCC (PD331) (A) or FANCD2 (PD20) (B) cells were mock treated or depleted for the indicated protein. Radials were analyzed with or without MMC treatment (40 ng/ml) as in Materials and Methods. Error bars represent standard error of the mean.

Fig. 7

Monoubiquitination of FANCD2 with depletion of Topo IIIα. Lanes 1 and 2, normal (GM639) cells with control siRNA; 3 and 4 with Topo IIIα siRNA. Cells were harvested 24 h after MMC treatment, as described in Materials and Methods.

Fig. 8

SCE formation in FA cells. FANCD2 (PD20) (A), FANCA (GM6914), FANCC (PD438F), FANCD1 (VU423), and FANCG (PD829) (B) were treated or not with MMC (20 ng/ml) and analyzed for SCE formation. Correction by complementing retroviral constructs is indicated, as is depletion by siRNA. Error bars represent standard error of the mean.