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. 2008 Dec;36(21):6907-17.
doi: 10.1093/nar/gkn793. Epub 2008 Oct 31.

Drosophila bloom helicase maintains genome integrity by inhibiting recombination between divergent DNA sequences

Michael Kappeler  1 , Elisabeth Kranz, Katrina Woolcock, Oleg Georgiev, Walter Schaffner
Affiliations

Drosophila bloom helicase maintains genome integrity by inhibiting recombination between divergent DNA sequences

Michael Kappeler et al. Nucleic Acids Res. 2008 Dec.

Abstract

DNA double strand breaks (DSB) can be repaired either via a sequence independent joining of DNA ends or via homologous recombination. We established a detection system in Drosophila melanogaster to investigate the impact of sequence constraints on the usage of the homology based DSB repair via single strand annealing (SSA), which leads to recombination between direct repeats with concomitant loss of one repeat copy. First of all, we find the SSA frequency to be inversely proportional to the spacer length between the repeats, for spacers up to 2.4 kb in length. We further show that SSA between divergent repeats (homeologous SSA) is suppressed in cell cultures and in vivo in a sensitive manner, recognizing sequence divergences smaller than 0.5%. Finally, we demonstrate that the suppression of homeologous SSA depends on the Bloom helicase (Blm), encoded by the Drosophila gene mus309. Suppression of homeologous recombination is a novel function of Blm in ensuring genomic integrity, not described to date in mammalian systems. Unexpectedly, distinct from its function in Saccharomyces cerevisiae, the mismatch repair factor Msh2 encoded by spel1 does not suppress homeologous SSA in Drosophila.

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Figures

Figure 1.
Figure 1.
Schematic representation of DNA constructs for SSA detection. (A) One of the two direct repeats, the one upstream of the I-SceI endonuclease cleavage site, is under the control of the mtnB enhancer/promoter. The coding region of the downstream repeat, lacking regulatory sequences for transcription, is fused in-frame to the EGFP cDNA. Induction of transcription does not lead to expression of EGFP protein, unless the DSB, generated by the endonuclease I-SceI, is repaired via SSA of the flanking metallothionein copies. (B) All constructs contain an I-SceI endonuclease recognition site (I-SceI), which is flanked by genomic (gen) or cDNA copies of mtnB. The length of sequence identity between tandem duplicates is indicated by blue bars and numbers, which represent nucleotide numbers. The longest stretch of sequence identity is 259 nt in the construct containing two genomic mtnB copies (MtnBgen-gen). This identity is disturbed in several constructs either by a silent point mutation (MtnBgen*-gen, MtnBcDNA-cDNA*) or the absence of the intron (MtnBcDNA-gen, MtnBgen-cDNA).
Figure 2.
Figure 2.
SSA recombination is impaired by mismatches and spacer DNA in transiently transfected S2 cells. S2 cells were transfected with two plasmids, one containing the SSA tester construct and the other encoding the endonuclease I-SceI under the actin promoter. SSA products resulted in EGFP cDNA driven by the copper inducible mtnB enhancer/promoter. 12 h after transfection EGFP expression was induced by addition of 250 μM CuSO4. (A) In order to reduce the background luminescence, green fluorescence was plotted against red. All cells with specific green fluorescence are shifted to the right. An area for EGFP positive cells was defined (G). For every sample the percentage of EGFP positive cells (nEGFP) and the mean fluorescence intensity of these cells (x̄EGFP) were determined. All indicated constructs are depicted in Figure 1B except MtnBgen-1000/0-gen, which contains an additional 1000 bp spacer upstream of the I-SceI recognition site. (B) On the x-axis the length of spacer sequences upstream/downstream of the I-SceI site are indicated. Green fluorescence as a measure for SSA was calculated as described in Materials and Methods section. Error bars represent standard deviations of three experiments.
Figure 3.
Figure 3.
Verification of SSA in transiently transfected S2 cells. (A) Constructs for transfection are depicted: enhancer/promoter-containing mtnB copy (MtnBgen) and enhancer/promoter-less mtnb-EGFP fusion copy (MtnBgen-EGFP). For the positive control construct for intramolecular SSA (MtnBgen-gen), see Figure 1B. Constructs indicated in the bar diagram were always cotransfected with a plasmid expressing the endonuclease I-SceI. Green fluorescence as a measure for SSA was calculated as described in Materials and Methods section. Error bars represent standard deviations of three experiments. (B) Isolation of SSA products from S2 cells. EGFP positive cells were first sorted, and plasmid DNA was isolated. PCR to detect SSA products was performed. The indicated tester constructs are depicted inFigure 1B. PCR products of plasmid DNA from EGFP positive S2 cells after DSB induction (S2) and from E. coli without induction of DSB (Ec) are shown. Expected PCR bands for SSA products are 431 bp and 370 bp (with and without the intron). The three marker bands shown indicate the position of 300 bp, 400 bp and 500 bp.
Figure 4.
Figure 4.
In vivo detection of SSA in the male germline. SSA frequency in flies was determined as described in Material and Methods section. Graphs show box plots for SSA frequencies with the middle line marking the median frequency of all tubes, boxes represent 25th and 75th percentiles and whiskers 10th and 90th percentiles. The two-tailed Mann Whitney U-Test was performed to assess statistical significance. **P < 0.01; ***P < 0.001. All constructs are depicted inFigure 1B. (A) SSA frequencies of MtnBgen-gen tester transgenes inserted at four different loci in the genome. The test lines were generated by targeted integration using the attP/attB system (phi) or random insertion using the traditional P-element system (p1, p2, p3). (B) SSA frequency of the MtnBgen-gen tester construct (with direct repeats spaced by 200 nt) compared to MtnBgen-gen containing an additional 2.2 kb spacer sequence between the repeats, downstream of the I-SceI recognition site. (C–F) SSA frequencies of the tester constructs indicated.
Figure 5.
Figure 5.
Homeologous SSA in male germline of blm (mus309) mutants and spel1 mutants. Box plots for SSA frequencies are depicted with the middle line marking the median of all tubes, boxes represent 25th and 75th percentiles and whiskers 10th and 90th percentiles. (A) Analysis of blm mutants. The rare cutting endonuclease I-SceI was expressed under control of the heat shock promoter in blm compound heterozygotes (mus309D3/mus309D2), in heterozygotes (mus309D3/+) and wt controls; they are labeled in the graph with −/−, +/− and +/+, respectively. All constructs (MtnBgen-gen, MtnBgen*-gen and MtnBcDNA-gen) are depicted inFigure 1B. SSA PCR products of all EGFP positive flies were sequenced to exclude random in-frame NHEJ events, graphs represent corrected data. The two-tailed Mann Whitney U-Test was performed to test for statistical significance between flies deficient (red) and proficient (black) for Blm. *P < 0.05; ***P < 0.001. In this experiment, tester constructs were inserted with the help of P-elements at different genomic loci on the second chromosome. SSA frequencies can therefore only be compared within the same tester construct. (B) Analysis of SSA in flies mutant for the msh2 homolog spel1. SSA was analyzed in spel1 compound heterozygotes (−/−) and heterozygous controls (+/−), which were both derived from a cross of spel1 heterozygous parents. The tester constructs MtnBgen-gen, MtnBgen*-gen and MtnBcDNA-gen (Figure 1B) are integrated at the attP landing site on the third chromosome, and MtnB-MtnD (Figure 6A) was P-element inserted on the X-chromosome (line px-3).
Figure 6.
Figure 6.
Homeologous SSA between two related genes is enhanced in blm mutant male germline. (A) Schematic representation of an SSA event between two metallothionein paralogs, mtnB (red) and mtnD (orange). Dotted blue line marks region of 80% sequence identity. (B) The frequency of SSA events between mtnB and mtnD in blm and spel1 mutants. The MtnB-MtnD construct tested here was inserted via P-elements on the X-chromosome (px-6). I-SceI was expressed under control of the heat shock promoter in blm mutant flies (mus309D3/mus309D2), as well as in controls (mus309D3/+ and +/+). In an additional experiment it was also expressed in a Spel1 deficient background. From the Blm experiment, SSA products of all EGFP positive offspring were sequenced. Identical SSA product sequences from the same fly tube were considered to originate from a single SSA event. Bars represent frequencies of distinguishable SSA events per total offspring flies analyzed, as indicated by numbers. The χ2-test was performed to test for statistical significance between blm mutant flies and controls. ***P < 0.001. (C) List of detected SSA products. The top two lines represent cDNA sequences of mtnB and mtnD. Divergent nucleotides are highlighted in red (mtnB) and orange (mtnD). All detected SSA events from blm mutant flies (−) and controls (+) are shown. DNA segments where recombination occurred are highlighted in grey.
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