The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeast dna2 mutants

Abstract

Bloom syndrome is a disorder of profound and early cancer predisposition in which cells become hypermutable, exhibit high frequency of sister chromatid exchanges, and show increased micronuclei. BLM, the gene mutated in Bloom syndrome, has been cloned previously, and the BLM protein is a member of the RecQ family of DNA helicases. Many lines of evidence suggest that BLM is involved either directly in DNA replication or in surveillance during DNA replication, but its specific roles remain unknown. Here we show that hBLM can suppress both the temperature-sensitive growth defect and the DNA damage sensitivity of the yeast DNA replication mutant dna2-1. The dna2-1 mutant is defective in a helicase-nuclease that is required either to coordinate with the crucial Saccharomyces cerevisiae (sc) FEN1 nuclease in Okazaki fragment maturation or to compensate for scFEN1 when its activity is impaired. We show that human BLM interacts with both scDna2 and scFEN1 by using coimmunoprecipitation from yeast extracts, suggesting that human BLM participates in the same steps of DNA replication or repair as scFEN1 and scDna2.

Fig. 1.

(A) hBLM and hDNA2 complement the dna2-1 temperature-sensitive phenotype. Strain dna2-1 was transformed with the following plasmids: empty vector (pRS316), pGAL-hBLM, pGAL-FLAG-mWRN, pGAL-FLAG-hRecQ4, pGAL-hDNA2-FLAG, and pGAL-xDNA2. Transformants were streaked onto SC galactose minus Ura and incubated at 23°C(Upper) and 37°C(Lower) for 10 days. (B) Analysis of expression levels for the hBLM and hDNA2 proteins in dna2-1 cells. Strains dna2-1 transformed with empty vector (pRS316), pGAL-hBLM, and pGAL-hDNA2-FLAG were grown in SC galactose minus Ura and harvested at the indicated times to prepare protein extracts. Extracts were prepared by the alkaline lysis method. Equal amounts of protein were loaded onto SDS/7.5% PAGE and immunoblots were probed with either anti-BLM (Upper) or anti-FLAG (Lower) antibodies. The positions of the hBLM and hDNA2 bands are indicated on the right. (C) Strain dna2-1 carrying empty vector (pRS316), pGAL-hBLM, pGAL-hDNA2-FLAG, or pGAL-xDNA2 was streaked onto SC galactose minus Ura plates with (Right) or without (Left) 5-FOA, and grown at 37°C for 7 days.

Fig. 2.

The helicase activity of hBLM is required for complementation of dna2-1. (A) hBLM helicase activity is required for complementation. The dna2-1 cells carrying empty vector (pRS316), pGAL-hBLM, or pGAL-hBLM(K695T) were streaked onto SC galactose minus Ura plates and grown at 23°C or 37°C for 7 days. The K695T mutation changes lysine to arginine at amino acid position 695 within the helicase domain of hBLM. (B) Expression of hBLM(K695T). Strain dna2-1 transformed with various vectors were analyzed by Western blotting with anti-hBLM antibody as described in Fig. 1B. Lane Vec, dna2-1 transformed with empty vector (pRS316); lane BLM, pGAL-hBLM; lane K695T, pGAL-hBLM(K695T). The position of hBLM is indicated. The helicase mutant protein always migrates slightly more slowly than the wild-type protein, as noted (9). (C) Complementation assay at different temperatures. Exponentially growing yeast cultures of dna2-1 cells carrying empty vector (pRS316), pGAL-hBLM, pGAL-hBLM(K695T), pGAL-FLAG-mWRN, pGAL-FLAG-hRecQ4, and pGAL-hDNA2-FLAG were spotted in 10-fold serial dilutions onto SC glucose minus Ura or SC galactose minus Ura plates and grown at 23°C, 35°C, and 37°C for 5–7 days.

Fig. 3.

Complementation of sensitivity of dna2-1 to MMS and HU. Exponentially growing yeast cultures of wild-type cells carrying empty vector (pRS316) and dna2-1 cells carrying empty vector, pGAL-hBLM, pGAL-hBLM(K695T), pGAL-FLAG-mWRN, pGAL-FLAG-RecQ4, and pGAL-hDNA2-FLAG were spotted in 10-fold serial dilutions onto SC galactose minus Ura plates with or without MMS or HU at the concentrations indicated and grown at the permissive temperature for 6 days. This experiment was performed in duplicate with identical results.

Fig. 4.

Lack of complementation of temperature-sensitive growth and MMS sensitivity of strain rad27Δ. Exponentially growing yeast cultures of W303 wild type, rad27Δ, rad27Δ/vector, rad27Δ/pGAL-hBLM, and rad27Δ/Yep24-Rad27 were spotted in 10-fold serial dilution onto plates. (A) Growth at 30°Cor37°C. (B) Growth in the presence and absence of 0.005% MMS. Cells were grown for 3 days.

Fig. 5.

Interaction between hBLM and replication fork proteins. (A) Interaction between hBLM and scFEN1. Extracts from BJ5459 transformed with pGAL-hBLM, pJDG-Rad27-myc encoding scFEN1-myc, or both were prepared, and immunoprecipitations were performed with either anti-myc (Upper) or anti-BLM (Lower) antibodies. Washed immunoprecipitates were loaded onto SDS/7.5% PAGE and immunoblotted with either anti-BLM or anti-myc antibodies, as indicated. (B) Interaction of the hBLM(K695T) mutant protein with scFEN1. Extracts were prepared from BJ5459 cells carrying pJDG-Rad27-myc and pGAL-hBLM or pGAL-BLM(K695T) and subjected to immunoprecipitation with anti-myc antibodies. The immunoprecipitates were immunoblotted with either anti-BLM or anti-myc antibodies, as indicated. BLM(K695T) always migrates slightly more slowly than wild-type BLM protein (see also Fig. 2B). (C) Interaction of hBLM with scDNA2. Extracts were prepared from BJ5459 cells carrying pGAL-hBLM and pJDG-DNA2-HA and subjected to immunoprecipitation with anti-HA antibodies. The immunoprecipitates were immunoblotted with either anti-BLM or anti-HA antibodies, as indicated. WCE, whole-cell extract before immunoprecipitation; IP, immunoprecipitation/immunoprecipitate; WB, Western blot of immunoprecipitate; -, control with no antibody in IP mix; +, presence of indicated antibody in IP mix.