Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms - PubMed (original) (raw)

Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms

S Lambert et al. EMBO J. 2000.

Abstract

In contrast to yeast RAD51, mammalian mRAD51 is an essential gene. Its role in double strand break (DSB) repair and its consequences on cell viability remain to be characterized precisely. Here, we used a hamster cell line carrying tandem repeat sequences with an I-SCE:I cleavage site. We characterized conservative recombination after I-SCE:I cleavage as gene conversion or intrachromatid crossing over associated with random reintegration of the excised reciprocal product. We identified two dominant-negative RAD51 forms that specifically inhibit conservative recombination: the yeast ScRAD51 or the yeast-mouse chimera SMRAD51. In contrast, the mouse MmRAD51 stimulates conservative recombination. None of these RAD51 forms affects non-conservative recombination or global DSB healing. Consistently, although resistance to gamma-rays remains unaffected, MmRAD51 stimulates whereas ScRAD51 or SMRAD51 prevents radiation-induced recombination. This suggests that mRAD51 does not significantly affect the global DSB repair efficiency but controls the classes of recombination events. Finally, both ScRAD51 and SMRAD51 drastically inhibit spontaneous recombination but not cell proliferation, showing that RAD51-dependent spontaneous and DSB-induced conservative recombination can be impaired significantly without affecting cell viability.

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Figures

Fig. 1. Overexpression of the different RAD51 genes in hamster cells. (A) Structure of the RAD51 genes used. The boxes correspond to the homologous region between the different RAD51 genes: grey, mouse MmRAD51; white, yeast ScRAD51. ScRAD51 is longer at the N-terminal part. The white box corresponds to the region homologous to MmRAD51 and the hatched box to the extra sequence. The numbers correspond to the positions of the amino acids. The numbers in black correspond to the yeast amino acids and the numbers in grey correspond to the mouse amino acids. The chimeric protein SMRad51 was constructed with the N-terminal part of ScRad51 (hatched box) fused to the entire MmRad51 (white box). ΔNtScRad51 corresponds to ScRad51 with 55 N-terminal amino acids deleted. These proteins are expressed in the CHO-DRA10 line, and the corresponding derivative lines are listed on the right. (B). Overexpression of exogenous RAD51. Protein extracts were obtained from stable transfectants. The expression of MmRAD51 and SMRAD51 was verified by western blotting using an antibody raised against the human Rad51 protein and normalized with an anti-actin antibody. Rm2 and 4 correspond to two independent stable transfectants overexpressing MmRAD51. SMRad51-1 and SMRad51-2 correspond to two independent stable transfectants overexpressing the fusion SMRAD51. Cm3 is a control clone corresponding to the CHO-DRA10 line transfected with the empty pCDNA3.1puro plasmid. Expression of ScRAD51 is visualized by western blotting using an anti-ScRad51 antibody. The first lane corresponds to transient expression (Tr) of ScRAD51 (used as control). The second lane corresponds to extracts from mock-transfected cells (CHO-DRA10). The third and fourth lanes correspond to two independent clones with stable expression of ScRAD51. MmRad51 is Rad51 from Mus musculus; ScRad51 is Rad51 from S.cerevisiae; CgRad51 is Rad51 from Cricetellus griseus; and SMrad51 is a chimeric fusion protein MmRad51–ScRad51.

Fig. 2. Strategy used to measure DSB-induced recombination events. After the formation of a DSB induced by I-_Sce_I, two processes can compete. (A) The non-conservative SSA that produces NeoR and hygromycin-sensitive recombinants. (B) Homologous recombination events leading to gene conversion either with or without crossing over. The products of gene conversion events are resistant to both G418 and hygromycin (NeoR–HygR). Intrachromatid crossing over leads to the formation of POCs. If the POCs are eliminated, the products are only NeoR; if the POC is reintegrated into the genome, it could result in a NeoR–HygR colony. Thus, double resistance (NeoR–HygR) scores only conservative recombination events. Not shown are unequal sister chromatid exchange events in which the HygR segregates from the NeoR gene.

Fig. 3. (A) Restriction pattern of recombinant clones induced by an I-_Sce_I-generated DSB. Probe A corresponds to the S2neo promoter sequence and probe B to part of the hygromycin-resistant gene, located between the two neo cassettes. The corresponding expected _Xho_I–_Hin_dIII sizes (with both probes) and the corresponding resistance phenotype are indicated. POC: pop out circle (see Figure 2). (B) Southern blot analysis with probe A. The sizes of the bands are indicated on the sides of the panels. Left panel: seven independent NeoR–HygR clones (numbered on the top of the panel) from the control cell line Cm3. Right panel: four independent NeoR–HygR clones (numbered on the top of the panel) from the Rm2 cell line, overexpressing MmRAD51. (C) The same filters as in (B) but hybridized to probe B, characteristic of the intervening sequence.

Fig. 4. Effect of the overexpression of MmRAD51, ScRAD51 or SMRAD51 on the frequency of conservative recombination events. Control refers to the parental line transfected with empty expression vector (Cm3). MmRAD51 refers to two independent transfectants overexpressing MmRAD51; ScRAD51 refers to one transfectant expressing yeast ScRAD51; SMRad51-1 and SMRad51-2 refer to two independent clones expressing the chimera SMRAD51; ScRad51ΔNt-1 and ScRad51ΔNt-23 refer to two independent clones expressing the deleted ΔNt_ScRAD51_. Values correspond to the percentage of double-resistant (NeoR–HygR) colonies (black boxes) and single-resistant (NeoR) colonies (grey boxes). The percentage of conservative recombination events is calculated from the ratio of the number of NeoR–HygR clones to the NeoR clone frequency. The percentages are indicated on the top of the corresponding histograms.

Fig. 5. Effect of RAD51 on radiation resistance (A) and on radiation-induced recombination (B). Cells were irradiated at the doses indicated. Controls correspond to the parental CHO-DRA10 line and to Cm3, which is CHO-DRA10 transfected with the empty expression vector. Rm2 and Rm4 are two independent clones overexpressing MmRAD51. ScRad51-3 and ScRad51-6 are two independent clones expressing ScRAD51. Radiation-induced recombination (B): the values correspond to the number of NeoR in 106 surviving irradiated cells, following subtraction of the number of NeoR in 106 non-irradiated cells.

Fig. 6. Effect of the overexpression of the different RAD51 forms on spontaneous recombination. (A) MmRAD51 stimulates the spontaneous recombination rate measured by fluctuation analysis using the Luria and Delbruck test (Luria and Delbruck, 1943). The names of the different lines are indicated under the histograms. DRA10, parental line; Cm3 (control line), DRA10 transfected with empty expression vector; Rm2 and Rm4 correspond to two independent clones overexpressing MmRAD51. The number of independent cultures for each line is reported under the histograms. (B) ScRAD51 and SMRAD51 inhibit the frequency of spontaneous recombination. A recombination rate cannot be calculated because most of the cultures do not contain any recombinant. The frequency of recombination reported here corresponds to the sum of the recombinants from all the independent cultures in relation to the total number of cells summed from all the independent cultures. DRA10 is the parental line; ScRad51-3 and ScRad51-6 correspond to two independent clones expressing ScRAD51; SMRad51-1, SMRad51-2 and SMRad51-24 refer to three independent clones expressing the chimera SMRAD51.

Fig. 7. Role of RAD51 in DSB repair in mammalian cells. On a DSB, Ku and RAD52 compete to process the DSB. Ku orientates DSB repair towards NHEJ. Conversely, RAD52 orientates repair to an HD process that can be either SSA or homologous recombination. In the absence of RAD51, SSA takes place. This is the most frequent reaction in the parental lines. If mRAD51 is present, it can bind to RAD52 and channel the reaction to homologous recombination initiated by strand invasion.

References

1. Arnaudeau C., Helleday,T. and Jenssen,D. (1999) The RAD51 protein supports homologous recombination by an exchange mechanism in mammalian cells. J. Mol. Biol., 289, 1231–1238. - PubMed
1. Barlow A.L., Benson,F.E., West,S.C. and Hulten,M.A. (1997) Distribution of the Rad51 recombinase in human and mouse spermatocytes. EMBO J., 16, 5207–5215. - PMC - PubMed
1. Baumann P. and West,S.C. (1998) Role of the human RAD51 protein in homologous recombination and double-stranded-break repair. Trends Biochem. Sci., 23, 247–251. - PubMed
1. Baumann P., Benson,F.E. and West,S.C. (1996) Human RAD51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell, 87, 757–766. - PubMed
1. Benson F.E., Baumann,P. and West,S.C. (1998) Synergistic actions of Rad51 and Rad52 in recombination and DNA repair. Nature, 391, 401–404. - PubMed

Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms - PubMed (original) (raw)