Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange - PubMed (original) (raw)

Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange

M L Dronkert et al. Mol Cell Biol. 2000 May.

Abstract

Cells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we analyzed the effect of mRAD54, a gene involved in homologous recombination, on the repair of a site-specific I-SceI-induced DSB located in a repeated DNA sequence in the genome of mouse embryonic stem cells. We used six isogenic cell lines differing solely in the orientation of the repeats. The combination of the three recombination-test substrates used discriminated among SSA, intrachromatid gene conversion, and sister chromatid gene conversion. DSB repair was most efficient for the substrate that allowed recovery of SSA events. Gene conversion with crossover, indistinguishable from long tract gene conversion, preferentially involved the sister chromatid rather than the repeat on the same chromatid. Comparing DSB repair in mRAD54 wild-type and knockout cells revealed direct evidence for a role of mRAD54 in DSB repair. The substrate measuring SSA showed an increased efficiency of DSB repair in the absence of mRAD54. The substrate measuring sister chromatid gene conversion showed a decrease in gene conversion with and without crossover. Consistent with this observation, DNA damage-induced sister chromatid exchange was reduced in mRAD54-deficient cells. Our results suggest that mRAD54 promotes gene conversion with predominant use of the sister chromatid as the repair template at the expense of error-prone SSA.

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Figures

FIG. 1

FIG. 1

Generation of mRAD54+/− and _mRAD54_−/− ES cells containing recombination-test substrates. (A) Structure of the genomic mRAD54 locus and targeting vectors containing the substrates. The two upper lines represent the wild-type (mRAD54+) and the puromycin-targeted knockout (mRAD54307pur) alleles, respectively. The 18 exons that encode mRad54 are indicated by boxes. The dashed line above exons 7 and 8 indicates the position of the probe used to distinguish the different mRAD54 alleles after digestion of the genomic DNA with _Stu_I. The arrow shows the position of the puromycin (pur) selectable marker gene. The locations of selected restriction sites are shown: E, _Eco_RI; N, _Nco_I; Sf, _Sfu_I; St, _Stu_I. The third line shows a generic representation of the targeting vectors. The three lower lines show the three different substrates inserted into the mRAD54 locus in more detail. The black arrow indicates the hygromycin (hyg)-selectable marker gene. The gray arrow on the left represents the 700-bp 3′ neomycin-selectable marker gene (3′ neo). The gray arrow on the right represents the full-length S2neo gene, which contains a 4-bp deletion at the 18-bp I-_Sce_I site insertion (indicated in black). (B) DNA blot of ES cells containing wild-type (+) and knockout (−) mRAD54 alleles in addition to alleles with recombination-test substrates. Genomic DNA was digested with _Stu_I. The DNA blot was hybridized with the probe indicated in panel A. Phage λ DNA digested with _Pst_I was used as a size marker. The lengths of marker fragments are indicated in kilobases on the right and the positions of the different mRAD54 alleles are shown on the left.

FIG. 2

FIG. 2

Model of possible mechanisms for homology-dependent DSB repair on DRneo. The DSB induced at the I-_Sce_I site and indicated by the gap in S2neo can be repaired by different repair pathways that are depicted schematically. Only repair events yielding an intact neo gene are shown. A summary of all possible outcomes of DSB repair is given in Table 1, and the different pathways are described in detail in the text. The annealing of the complementary ssDNA during SSA is indicated by thin vertical lines. Pairing of S2neo and 3′ neo (indicated by the cross) can result in GC with or without CO. Symbols are the same as those in Fig. 1.

FIG. 3

FIG. 3

DNA blot analysis of I-_Sce_I-induced DSB repair events in ES cells containing the different recombination-test substrates. _mRAD54_-proficient ES cells containing either DRneo, IRneo, or SCneo were transfected with an I-_Sce_I-expressing plasmid. After selection with G418 or G418-hygro, genomic DNA from individual clones was digested with _Eco_RI. The outcome of repair of the I-_Sce_I-induced DSB was analyzed by DNA blotting using a 700-bp 3′ neo probe. Only a selection of the clones listed in Table 2 is shown. As shown in Fig. 2 and 5, the sizes of the _Eco_RI fragments labeled with the neo probe indicate whether the DSB has been repaired by GC or CO. With DRneo, SSA results in the same molecular outcome as CO. Phage λ DNA digested with _Pst_I was used as a size marker. The lengths of marker fragments are on the left.

FIG. 4

FIG. 4

Homologous recombination frequencies for the recombination-test substrates. As described in Materials and Methods, 1.6 × 106 _mRAD54_-proficient and -deficient ES cells containing the indicated substrates in the identical genomic location were transfected with pCBA3xnls-I-_Sce_I and processed. Shown is the normalized number of G418- or G418-hygro-resistant colonies ± standard error of the mean for three independent experiments with two cell lines from all six genotypes. (A) HR frequency of mRAD54 +/DRneo (+/−) and mRAD54−/DRneo (−/−) ES cells. Colonies containing an intact neo gene were obtained after repair of the I-_Sce_I-induced DSB by SSA, GC, and CO. (B) Frequencies of GC and CO for ES cells containing the substrates. For all three substrates, _neo_- and _hyg_-containing colonies were obtained after repair of the I-_Sce_I-induced DSB by intrachromatid GC and sister chromatid GC. The IRneo- and SCneo-containing cell lines each have one additional possibility to yield G418-hygro-resistant colonies. In the IRneo-containing lines, these clones can be formed by intrachromatid CO. For the SCneo-containing lines, they can be formed by CO after pairing with the sister chromatid.

FIG. 5

FIG. 5

Schematic representation of possible homology-dependent DSB repair pathways for IRneo and SCneo. Only repair events yielding an intact neo gene are depicted. A summary of all possible outcomes of DSB repair is given in Table 1, and the different pathways are described in detail in the text. Symbols are the same as in Fig. 1. The I-_Sce_I-induced DSB is indicated by the gap in S2neo. Recombination between S2neo and 3′ neo, indicated by the cross, can lead to restoration of the original _Nco_I site resulting in an intact neo gene by GC with or without CO. Concerning the COs, only the product that results in an intact neo gene is shown. Shown are the outcomes of DSB repair events on IRneo (A) and SCneo (B).

FIG. 6

FIG. 6

Induction of SCEs by MMC in _mRAD54_-proficient and -deficient ES cells. ES cells of the indicated genotypes were either mock treated or treated with 0.2 μg of MMC/ml for 1 to 2 h, and metaphase spreads were prepared. Forty to 95 metaphases per sample were scored for the number of SCEs per cell. The frequency of spontaneous SCEs is shown in black, while the frequency of SCEs after treatment with MMC is shown in white. The error bars indicate the 95% confidence intervals.

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