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

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 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

Method for identifying non-canonical HR termination products in mammalian cells.

<p>(A) Schematic of the HR reporter. Duplication of a blasticidin resistance cassette during LTGC allows expression of wt <i>BsdR</i> by splicing. Thus, I-SceI-induced STGCs are GFP⁺ and Bsd sensitive (BsdR^–), while I-SceI-induced LTGCs are GFP⁺ and Bsd resistant (BsdR⁺). Most LTGCs resolve as “<i>GFP</i> triplication” events, but a small fraction of LTGCs resolve by non-canonical mechanisms. Non-canonical LTGC termination products can be distinguished by the structure of the LTGC product, as shown. (B) Characterization of <i>XRCC4</i>^fl/fl and <i>XRCC4</i>^Δ/Δ Cre-treated HR reporter clones. Upper panel: Southern blotting, as described in Materials and Methods. Lower panel: western blotting for XRCC4 or for ß-tubulin loading control.</p

MMBIR model of complex breakpoint shown in Fig 5B.

<p>Strand separation occurs within the DNA of the second end of the break ~3.5 kb from the I-SceI site. One possible source depicted here is a stalled replication fork. The pale orange and blue arrows flanking the stalled fork represent the exposed ssDNA sequences that template the inversion (orange) and inversion-duplication (blue) sequences identified within the LTGC breakpoint (A) The displaced nascent strand product of LTGC (black) acquires a ≥21bp insertion (red; whether templated or untemplated is unknown). (B) Microhomology-mediated base-pairing between the 3’ end of the displaced nascent strand and ssDNA of the stalled replication fork. (C) The lagging strand template enables retrograde nascent strand extension (“MMBIR”), generating the inversion sequences as shown. (D) Displacement of the nascent strand. (E) Four base pair MH-mediated (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006410#pgen.1006410.g006" target="_blank">Fig 6B</a>) annealing of the 3’ end of the displaced nascent strand with the 5’ end of the duplicated region on the leading strand. Black arrowheads: sites of endonucleolytic cleavage that would enable completion of rearrangement by MMEJ-mediated rejoining. Alternatively, more extensive MMBIR copying could complete the rearrangement.</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 an aberrant LTGC rearrangement in <i>XRCC4</i>^Δ/Δ HR/SCR reporter cells.

<p>(A) Restriction analysis of aberrant LTGC product in <i>XRCC4</i>^Δ/Δ HR/SCR reporter cells. Genomic DNA 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). Patterns suggest that LTGC was terminated by non-canonical mechanisms. Blue arrow-heads: deduced products of non-canonical LTGC termination. Blue star: off-size restriction fragments of HindIII, EcoRI and SpeI digests are inconsistent with rejoining with the second end of the original I-SceI-induced DSB (compare with <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006410#pgen.1006410.g002" target="_blank">Fig 2</a>). Orange arrow-heads: fainter bands may represent the half-copy of <i>GFP</i> retained by the second end of the original I-SceI-induced DSB. Note that the upper band of SpeI-restricted gDNA has a greater intensity than other bands, suggesting presence of two distinct co-migrating <i>GFP</i>-hybridizing fragments, or a single fragment containing >1 copy of <i>GFP</i>. (B) Deduced rearrangement of the non-canonically-terminated LTGC. Blue star: off-size restriction fragments of HindIII, EcoRI and SpeI digests. Note that each of these off-size fragments spans the predicted breakpoint of LTGC termination. This suggests that this LTGC event terminated by rejoining to ectopic chromosomal sequences (grey bar in the figure). (C) Deduced rearrangement of the second end of the DSB. Note that <i>GFP</i>-hybridizing fragments of SpeI, BamHI and HindIII restriction digest are off-size, potentially consistent with rejoining of the second end of the DSB with ectopic chromosomal sequences (grey bar).</p