Cell Cycle-Dependent Induction of Homologous Recombination by a Tightly Regulated I-SceI Fusion Protein

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase

A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

We have identified a novel gene in a genome-wide, double-strand break DNA repair RNAi screen and show that is involved in the neurological disease hereditary spastic paraplegia

Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells

A proportion of homologous recombination (HR) events in mammalian cells resolve by “long tract” gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer

Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells.

A proportion of homologous recombination (HR) events in mammalian cells resolve by "long tract" gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer

Breakpoints of non-canonical LTGC termination in five <i>XRCC4</i>^fl/fl and two <i>XRCC4</i>^Δ/Δ clones.

<p>Cartoon shows approximate positions of breakpoints. Black numbers mark site of LTGC termination; paired blue numbers mark extent of second end resection for the same clone (not to scale). Numbers correlate with the numbered clones in lower panel, showing length of gene conversion tract (black) and extent of second end resection (blue) in each clone, with genotype as indicated. Red nucleotides: N-insertions at the breakpoint. Dual black/blue nucleotide sequences at the breakpoint represent microhomology.</p

Restriction mapping of products of non-canonical LTGC termination.

<p>Genomic DNA from two clones in which LTGC was terminated by non-canonical mechanisms was digested with the restriction enzymes shown and analyzed by Southern blotting (<i>GFP</i> probe). Restriction enzymes used were SacI (Sa), HindIII (H), BamHI (B), EcoRI (E) and SpeI (Sp). Cartoons on right show restriction fragment sizes observed for HR reporter at the <i>ROSA26</i> locus. The presence or absence of the 3.2kb amplification product in each restriction digest helps to localize the site of LTGC termination within the reporter. (A) <i>XRCC4</i>^fl/fl clone in which termination of LTGC occurred between HindIII and EcoRI sites within the HR reporter. EcoRI and SpeI digests lack the 3.2kb amplification product. (B) <i>XRCC4</i>^Δ/Δ clone in which termination of LTGC occurred between SacI and HindIII sites within the HR reporter. HindIII, EcoRI and SpeI digests lack the 3.2kb amplification product. In this clone, the right hand arms of the SpeI and HindIII digests are much smaller (SpeI) or larger (HindIII) than would be predicted. This is explained by the deletion of ~3.5kb from the second end of the DSB, as revealed by sequencing (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006410#pgen.1006410.g006" target="_blank">Fig 6B</a>).</p

I-SceI-induced LTGC products in <i>XRCC4</i>^fl/fl and <i>XRCC4</i>^Δ/Δ cells.

<p>I-SceI-induced LTGC products in <i>XRCC4</i>^fl/fl and <i>XRCC4</i>^Δ/Δ cells.</p

Restriction mapping of parental reporter and of LTGC “<i>GFP</i> triplication” products.

<p>(A) Expected <i>GFP</i>-hybridizing gDNA restriction fragment sizes for HR reporter at the <i>ROSA26</i> locus. Upper panel: parental reporter; lower panel: “<i>GFP</i> triplication” outcome of LTGC. <i>GFP</i> copies within the reporter are shown. Filled ovals: artificial <i>BsdR</i> exons A and B. Restriction enzyme sites shown are SpeI (Sp), EcoRI (E), BamHI (B), HindIII (H) and SacI (Sa). Note that each of these restriction endonucleases, which cut target sites between the two <i>GFP</i> copies within the parental reporter, generate an additional 3.2kb <i>GFP</i>-hybridizing band in the context of the “<i>GFP</i> triplication” outcome. (B) Genomic DNA from parental and “<i>GFP</i> triplication” LTGC clones, as shown, was digested with the restriction enzymes shown (code as described above) and analyzed by Southern blotting (<i>GFP</i> probe). The 3.2kb amplification product characteristic of the “<i>GFP</i> triplication” LTGC outcome is marked with an arrowhead.</p