Functional interactions between BLM and XRCC3 in the cell

Abstract

Bloom's syndrome (BS), which is caused by mutations in the BLM gene, is characterized by a predisposition to a wide variety of cancers. BS cells exhibit elevated frequencies of sister chromatid exchanges (SCEs), interchanges between homologous chromosomes (mitotic chiasmata), and sensitivity to several DNA-damaging agents. To address the mechanism that confers these phenotypes in BS cells, we characterize a series of double and triple mutants with mutations in BLM and in other genes involved in repair pathways. We found that XRCC3 activity generates substrates that cause the elevated SCE in blm cells and that BLM with DNA topoisomerase IIIalpha suppresses the formation of SCE. In addition, XRCC3 activity also generates the ultraviolet (UV)- and methyl methanesulfonate (MMS)-induced mitotic chiasmata. Moreover, disruption of XRCC3 suppresses MMS and UV sensitivity and the MMS- and UV-induced chromosomal aberrations of blm cells, indicating that BLM acts downstream of XRCC3.

Figure 1.

Generation of double and triple mutants of BLM with mutations in other genes involved in DNA repair pathways. (A) Schematic representation of the generation of several mutants in a conditional xrcc3 background. (B) RT-PCR analysis of total RNA from the indicated mutants in the conditional xrcc3 background. (a–c) Each mutant cell line was examined for the expression of hXRCC3, BLM, RAD54, and RECQL1 mRNA. RECQL1 was amplified as a control. (d and e) Each mutant cell line was examined for the expression of hXRCC3, WRN, BLM, and RECQL1 mRNA. (C) Disruption of BLM in the indicated single gene mutants. (a) Schematic representation of the generation of mutants. (b–d) RT-PCR analysis of total RNA from the indicated mutants.

Figure 2.

Spontaneous SCE levels of various mutants. (A) SCE levels of xrcc3+hXRCC3 (“wild-type”), xrcc3/blm+hXRCC3 (“blm”), xrcc3, and xrcc3/blm cells. Numbers represent means and SDs of scores from two hundred metaphase cells. (B) SCE levels of xrcc3+hXRCC3, xrcc3/blm+hXRCC3, xrcc3/rad54+hXRCC3 (“rad54”), and xrcc3/blm/rad54+hXRCC3 (“blm/rad54”) cells. (C) SCE levels of wild-type, blm, rad52, and rad52/blm cells. (D) SCE levels of top3α and top3α/blm cells in a conditional top3α background. The expression of mouse Top3α is suppressed by doxycyclin. (E) SCE levels of wild-type, blm, top3β, and top3β/blm cells. The error bars show SD of scores from 100 metaphase cells.

Figure 3.

Survival curves of mutant cells exposed to MMS. (A–C) Cells were treated with the indicated concentrations of MMS. Colonies were counted after 7–14 d, and the percent survival was determined relative to the number of colonies of untreated cells. Representative data are shown. The error bars indicate SD. The differences in the duplicated data were often so minute that the SD was hidden by the symbols in the figures.

Figure 4.

MMS-induced chromosomal aberrations in mutant cells. (A and B) Chromosomal aberrations. Cells were cultured in the presence of 8 × 10⁻⁶ (vol/vol) MMS for 4 h and transferred to fresh medium without MMS. Cells were harvested at the indicated times, and 200 cells in the first metaphase were analyzed for chromosomal aberrations, as described in Materials and methods. (−) indicates spontaneous chromosomal aberrations. Data represents SEM. (A) xrcc3+hXRCC3 (“wild-type”), xrcc3/blm+hXRCC3 (“blm”), xrcc3, and xrcc3/blm cells. (B) Wild-type, blm, rad52, and rad52/blm cells. (C and D) MMS-induced mitotic chiasmata. Cells were treated as described in A and B. (C) Images of MMS-induced mitotic chiasma in cells 12 h after exposure to MMS. (a) Schematic of a mitotic chiasma. (b–f) Typical examples of mitotic chiasmata resulting from recombination between homologous chromosomes. The point of recombination between homologous chromosomes is indicated with an arrow. The numbers in the figures indicate the number of macrochromosomes. (D) 400 (a) and 200 (b) cells in the first metaphase 12 h after MMS exposure were analyzed. Data represents SEM. (a) xrcc3+hXRCC3, xrcc3/blm+hXRCC3, xrcc3, and xrcc3/blm cells. There was a statistically significant difference between xrcc3+hXRCC3 and xrcc3/blm+hXRCC3 cells (t test; *, P < 0.05). (b) Wild-type, blm, rad52, and rad52/blm cells.

Figure 5.

Suppression of UV sensitivity and UV-induced chromosomal aberrations in blm cells by disruption of XRCC3. (A) X-ray and UV sensitivities of xrcc3+hXRCC3 (“wild-type”), xrcc3/blm+hXRCC3 (“blm”), xrcc3, xrcc3/blm, and atm cells (Takao et al., 1999). Cells were irradiated with the indicated dose of x ray or UV. Colonies were counted after 10 d, and the percent survival was determined relative to the number of colonies derived from untreated cells. Representative data are shown. The error bars indicate SD. The differences in the duplicated data were often so minute that the SD were hidden by the symbols in the figures. X-ray (a) and UV survival curves (b) are shown. (B) UV-induced chromosomal aberrations (a) and mitotic chiasmata (b) in wild-type and blm cells. Samples were prepared every 3 h after irradiation with 8 J/m² UV. 200 cells in the first metaphase after UV irradiation were analyzed. Data represents the SEM. (C) UV-induced chromosomal aberrations (a) and mitotic chiasmata (b) in xrcc3+hXRCC3, xrcc3/blm+hXRCC3, xrcc3, and xrcc3/blm cells. Samples were prepared 12 h after irradiation with 8 J/m² of UV. 200 (a) and 400 (b) cells in the first metaphase were analyzed. (−) indicates spontaneous chromosomal aberrations. Data represents the SEM. There was a statistically significant difference between xrcc3+hXRCC3 and xrcc3/blm+hXRCC3 cells (t test; *, P < 0.05).

Figure 6.

Model of a role for BLM in the XRCC3-related damage tolerance pathway. (A) A model based on canonical recombination pathways. (B) A model for a damage tolerance pathway involving XRCC3 and BLM based on the S. cerevisiae model proposed by Liberi et al. (2005) and Branzei et al. (2006).