XRCC3 promotes homology-directed repair of DNA damage in mammalian cells - PubMed (original) (raw)

XRCC3 promotes homology-directed repair of DNA damage in mammalian cells

A J Pierce et al. Genes Dev. 1999.

Abstract

Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.

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Figures

Figure 1

Figure 1

GFP expression plasmids. (A) Modified GFP gene. The modified GFP gene encodes the EGFP protein fused to a nuclear localization signal (N) and zinc finger domain (Z). It is expressed from a hCMV enhancer/chicken β-actin promoter (arrow) on a spliced message (not shown). GFP is modified to SceGFP so as to contain an I-_Sce_I site and in-frame termination codons (underline). (B) DR–GFP recombination substrate. Downstream of the SceGFP gene is iGFP, a 5′ and 3′-truncated GFP gene. (C) (STGC) product. In a STGC, a DSB at the I-Sce I site is repaired from the iGFP gene on the same chromatid or sister chromatid, to result in a functional GFP gene. (D) Homology-mediated deletion product.

Figure 2

Figure 2

The irs1SF cell line is defective for DSB-induced homologous recombination because of XRCC3 deficiency. Wild-type (DRaa) and XRCC3-deficient (DRirs) cell lines were electroporated with each of the indicated expression vectors (100 μg of the I-Sce I vector; 20 μg of the XRCC3 vector), and 5 × 104 cells were analyzed by flow cytometry. (A) Two-color fluorescence analysis for cell lines DRaa-40 and DRirs-10. The percentage of green fluorescent cells falling above the diagonal for each transfection are indicated. (FL1) Green fluorescence; (FL2) orange fluorescence. (B) Summary of results from several cell lines.

Figure 3

Figure 3

Green fluorescence is stably expressed after I-Sce I expression. Wild-type DRaa-40 cells and XRCC3-deficient DRirs-14 cells were electroporated with the I-Sce I expression vector or both the I-Sce I and XRCC3 expression vectors, sorted on the basis of green fluorescence, and then reanalyzed by flow cytometry. Percent GFP+ cells before sorting (unshaded peak) and after sorting (shaded peaks) are indicated. Not shown is the DRaa-40 cell line transfected with both the I-Sce I and XRCC3 expression vectors, in which the presorted population was 0.66% GFP+ and the post-sorted population was 93.5% GFP+. Also not shown are the _lacZ_-transfected cells that were not sorted; 0.01% GFP+ for DRaa-40 and <0.01% GFP+ for DRirs-14. (The frequency of GFP+ cells is lower in this experiment than the one shown in Fig. 2, because only 20 μg of each of the expression vectors was electroporated.)

Figure 4

Figure 4

Green fluorescence is a measure of STGC. (A) Map of the parental or recombined DR–GFP substrate containing SceGFP or GFP, respectively. Sizes of expected fragments are indicated in base pairs. (B) Southern blot analysis of sorted cell lines. Genomic DNA was digested with the indicated endonucleases. (Lanes 1,2,5,6) Untransfected wild-type cell line DRaa-40; (lanes 3,7) GFP+ DRaa-40 cells from transfection of the I-Sce I expression vector; (lanes 4,8) GFP+ DRaa-40 cells from cotransfection of the I-Sce I and XRCC3 expression vectors; (lanes 9,10,13,14) untransfected mutant cell line DRirs-14; (lanes 11,15) GFP+ DRirs-14 cells from transfection of the I-Sce I expression vector; (lanes 12,16) GFP+ DRirs-14 cells from cotransfection of the I-Sce I and XRCC3 expression vectors. The residual 3021-bp band after _Bcg_I digestion in lanes 7,8,15, and 16 is likely due to GFP-negative cells in the population (see Fig. 3).

Figure 5

Figure 5

DSB-induced recombination is restored in the irs1SF cell line by transient expression of XRCC3 but not other recombination proteins. The DRirs-14 cell line was electroporated with each of the indicated expression vectors (20 μg). The total amount of DNA electroporated was maintained at a constant level by the addition of pCMV–lacZ. A total of 5 × 105 cells were analyzed by flow cytometry. (hRad51 or 51) Human Rad51; (mRad52 or 52) mouse Rad52; (X2) XRCC2; (X3) XRCC3.

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