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. 2000 Jul 3;19(13):3428-35.
doi: 10.1093/emboj/19.13.3428.

Possible association of BLM in decreasing DNA double strand breaks during DNA replication

W Wang  1 , M Seki, Y Narita, E Sonoda, S Takeda, K Yamada, T Masuko, T Katada, T Enomoto
Affiliations

Possible association of BLM in decreasing DNA double strand breaks during DNA replication

W Wang et al. EMBO J. .

Abstract

Bloom's syndrome (BS) is a rare genetic disorder and the cells from BS patients show genomic instability and an increased level of sister chromatid exchange (SCE). We generated BLM(-/-) and BLM(-/-)/RAD54(-/-) DT40 cells from the chicken B-lymphocyte line DT40. The BLM(-/-) DT40 cells showed higher sensitivity to methyl methanesulfonate and elevated levels of SCE as expected. The targeted integration frequency was also increased remarkably in BLM(-/-) cells. The SCE frequency increase in BLM(-/-) cells was considerably reduced and the enhanced targeted integration observed in BLM(-/-) cells was almost completely abolished in BLM(-/-)/RAD54(-/-) cells, indicating that a large portion of the SCE in BLM(-/-) cells occurs via homologous recombination, and homologous recombination events increase with the defect of BLM function. The BLM(-/-)/RAD54(-/-) cells showed a slow growth phenotype and an increased incidence of chromosome-type breaks/gaps while each single mutant showed relatively small numbers of chromosome-type breaks/gaps.

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Figures

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Fig. 1. Amino acid alignments in the helicase domain of chicken BLM (GdBLM) and human (Hs) BLM, WRN, RecQL1, RecQL4 and RecQL5. Thick lines indicate helicase motifs.
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Fig. 2. Generation of BLM–/– clones. (A) Schematic representation of disruption constructs. Motif III in chicken BLM genomic DNA cloned in a plasmid was replaced with a BamHI site by PCR, and his or bsr or hyg was inserted at this site. The regions of the probe used for Southern blot analysis are indicated. (B) Southern blot analysis. BamHI-digested genomic DNA prepared from wild-type, BLM+/– or BLM–/– cells was hybridized with the probes shown in (A). Lane 1, wild-type cells (+/+); lane 2, BLM+/– cells (+/–); lane 3, BLM–/– cells (–/–). (C) Northern blot analysis. Total RNA from wild-type or BLM–/– cells was hybridized with the chicken N- or C-terminal BLM cDNA probe synthesized by PCR as described in Materials and methods. The same filter was rehybridized with a chicken β-actin probe.
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Fig. 3. Growth curves of cells with various genotypes. Cells were inoculated into 3-cm dishes, and enumerated at the time indicated. The data show the average of the results from three independent experiments.
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Fig. 4. Sensitivity of cells with various genotypes to MMS, VP16 and CAM. Cells were treated with the indicated concentration of MMS (A), VP16 (B) or CAM (C) as described in Materials and methods. (A) The concentration of undiluted MMS (99%) is 1. The data show the average of the results from three independent experiments.
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Fig. 5. Histograms of SCE in wild-type, RAD54–/–, BLM–/– and BLM–/–/RAD54–/– cells. SCEs in the macrochromosomes were counted. Histograms show the frequency of cells with the indicated number of SCEs per cell. The mean number of SCEs per cell is shown in the upper right corner. (A) Wild-type cells; (B) BLM–/– cells; (C) BLM–/–/RAD54–/– cells; (D) RAD54–/– cells; (E) a superimposed figure of (A), (B), (C) and (D) expressed on the same scale.
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Fig. 6. Southern blot analysis of drug-resistant clones. (A) Schematic representation of the wild-type allele and the targeted allele. The dotted line corresponds to the targeting construct. (B) The DNA samples were digested with BamHI and hybridized to the probe shown in (A). A typical Southern blot is shown. Arrowheads indicate the position of the fragment representing the targeted allele. +, targeted integration.
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Fig. 7. Cell cycle analysis by flow cytometry. Distribution patterns of asynchronous culture of wild-type, BLM–/–, RAD54–/– and BLM–/–/RAD54–/– cells in the cell cycle.
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