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Results: 3

1.
Fig. 3.

Fig. 3. From: Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions.

HDR and ssDNA binding. (a) Interaction of BRC-RPA fusion proteins with the other RPA subunits. Whole-cell extracts from cells stably expressing BRC3RPA or BRC3ΔRPA were probed after gel electrophoresis either directly with α-RPA70, RPA32, and RPA14 antibodies or after immunoprecipitation with an α-FLAG antibody. Purified heterotrimeric RPA and whole-cell extracts from MCF-7 cells also are included. The weak band below the BRC-RPA proteins in the IP lanes is likely a degradation product that runs slightly above the position of RPA70 itself. (b) RPA mutations that reduce (K263A) or abolish (R234A/K263A) ssDNA-binding interfere with the ability of the BRC-RPA proteins to function in HDR. Asterisk indicates a statistically significant difference for transfection of the wild-type BRC3RPA expression vector compared with the other BRC3RPA expression vectors (K263A, P = 0.041 and R234A/K263A, P = 0.031; n = 3). (c) HDR is increased in V-C8 cells with transient expression of a Brh2-Rad52 fusion protein. The Brh2-Rad52 fusion contains the BRC repeat from U. maydis Brh2 with surrounding sequences (amino acids 89–551) fused to the conserved ssDNA binding domain of U. maydis Rad52 (amino acids 79–314); Brh2 extends from amino acids 89–955 and, hence, is truncated in the DNA-binding domain (18). Asterisks indicate a statistically significant difference from transfection with the empty expression vector (BRC3RPA, P = 0.0022 and Brh2-Rad52, P = 0.0027; n = 3).

Hiroshi Saeki, et al. Proc Natl Acad Sci U S A. 2006 June 6;103(23):8768-8773.
2.
Fig. 2.

Fig. 2. From: Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions.

Correction of the HDR defect and phenotypes associated with impaired HDR in Brca2 mutant cells by stable expression of the BRC-RPA proteins. (a) BRC-RPA expression vectors were cotransfected into V-C8 cells with a neomycin phosphotransferase gene (neo+). Immunoprecipitation with anti-FLAG antibody followed by Western blot analysis shows BRC-RPA expression (α-FLAG) and interaction with Rad51 (α-Rad51). (b) HDR is increased nearly to wild-type levels in V-C8 cells stably expressing BRC-RPA proteins. Asterisks indicate a statistically significant difference from parental V-C8 cells (P < 0.0001; n = 6). (c) SSA is suppressed in V-C8 cells stably expressing BRC-RPA proteins. The 0.8-kb PCR fragment derived from primers SA-F and SA-R2 specifically detects the SSA repair product, whereas the 1.1-kb PCR fragment from primers SA-F and SA-R1 detects a structurally intact reporter, i.e., from HDR and NHEJ, as well as the parental DR-GFP reporter. See Fig. 5a for quantitation. (d) Rad51 focus formation in response to DNA-damaging agents is restored in V-C8 cells stably expressing BRC-RPA proteins. Representative wild-type (V79), Brca2 mutant (V-C8), or Brca2 mutant cells stably expressing the indicated BRC-RPA peptides are shown after IR. Note that Rad51 is diffusely present on the chromatin of the BRC3RPA and BRC1–4RPA cell lines. Exponentially growing cells were irradiated with 12 Gy of IR and analyzed 7 h later for Rad51 foci. See Fig. 5b for quantitation. (e) Hypersensitivity of Brca2 mutant cells to DNA-damaging agents is reduced or eliminated with BRC-RPA expression. Survival was assessed by crystal violet staining after treatment for 24 h (MMC) or by clonogenic survival (IR). The percent survival of cells treated with DNA-damaging agents was computed relative to that of untreated cells, which was set to 100% for each line. Each percentage shown is the mean and error bars represent the SDs. MMC treatments were triplicated except that BRC3RPA and BRC1-2RPA stable cell lines were treated six times. IR treatment for each dose was performed once, except that 6-Gy treatments were quadruplicated.

Hiroshi Saeki, et al. Proc Natl Acad Sci U S A. 2006 June 6;103(23):8768-8773.
3.
Fig. 1.

Fig. 1. From: Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions.

Transient expression of BRC-RPA fusion proteins increases HDR in Brca2 mutant cells. (a) Human BRCA2. BRCA2 has a central region containing eight BRC repeats which bind Rad51. C-terminal to the BRC repeats is a region of higher conservation that encompasses a DNA-binding domain (DBD). A distinct Rad51-binding motif is found at the C terminus. (b) BRC-RPA fusion proteins and BRC repeat peptides. RPA70 refers to the entire human RPA70-coding sequence. BRC3Δ contains a 7-aa deletion that abrogates Rad51 binding. A nuclear localization signal (nls) is present at the N terminus and a FLAG epitope tag at the C terminus. (c) Western blot analysis of Brca2 mutant V-C8 hamster cells transiently expressing the BRC-RPA fusion proteins and BRC repeat peptides. Sizes of the expressed peptides are as expected. Asterisks denote a background immunoreactive band. (d) Flow cytometric analysis of V-C8 cells demonstrates increased HDR after transient expression of the BRC3RPA fusion protein as compared with BRC3ΔRPA. V-C8 cells containing a chromosomal DR-GFP reporter were cotransfected with expression vectors for the I-SceI endonuclease and the indicated BRC-RPA fusion protein. Cleavage of the DR-GFP reporter at the I-SceI site in vivo at the SceGFP gene and repair by HDR directed by the downstream iGFP repeat results in GFP-positive cells. (e) HDR is increased in V-C8 cells with transient expression of the BRC-RPA fusion proteins but not with the BRC repeat peptides. Asterisks indicate a statistically significant difference from transfection with the empty expression vector by using an unpaired t test (BRC3RPA, P = 0.013; BRC1–2RPA, P = 0.0004; BRC1–4RPA, P = 0.0048; BRC4RPA, P < 0.0001) (see also Fig. 4a). (f) HDR in wild-type V79 hamster cells with expression of the BRC peptides or the BRC-RPA fusion proteins. Asterisks indicate a statistically significant difference from transfection with the empty expression vector (BRC3, P = 0.0002; BRC1-2, P = 0.0006; BRC1-4, P = 0.0007) (see also Fig. 4b). (g) HDR in CAPAN-1 cells is increased by transient expression of BRC1-2RPA. The human pancreatic adenocarcinoma cell line CAPAN-1 carries a 6174delT mutation on one allele of BRCA2 with loss of the other wild-type BRCA2 allele. Asterisk indicates a statistically significant difference in HDR between transfection of the BRC1-2RPA expression vector and the empty expression vector (P < 0.05). Error bars in eg indicate 1 SD from the mean. Results are derived from three independent transfections for each sample (n = 3).

Hiroshi Saeki, et al. Proc Natl Acad Sci U S A. 2006 June 6;103(23):8768-8773.

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