Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/<i>Ter</i>-mediated replication fork barrier

<p>The <i>Escherichia coli</i> replication fork arrest complex Tus/<i>Ter</i> mediates site-specific replication fork arrest and homologous recombination (HR) on a mammalian chromosome, inducing both conservative “short tract” gene conversion (STGC) and error-prone “long tract” gene conversion (LTGC) products. We showed previously that bidirectional fork arrest is required for the generation of STGC products at Tus/<i>Ter</i>-stalled replication forks and that the HR mediators BRCA1, BRCA2 and Rad51 mediate STGC but suppress LTGC at Tus/<i>Ter</i>-arrested forks. Here, we report the impact of <i>Ter</i> array length on Tus/<i>Ter</i>-induced HR, comparing HR reporters containing arrays of 6, 9, 15 or 21 <i>Ter</i> sites—each targeted to the <i>ROSA26</i> locus of mouse embryonic stem (ES) cells. Increasing <i>Ter</i> copy number within the array beyond 6 did not affect the magnitude of Tus/<i>Ter</i>-induced HR but biased HR in favor of LTGC. A “lock”-defective Tus mutant, F140A, known to exhibit higher affinity than wild type (wt)Tus for duplex <i>Ter</i>, reproduced these effects. In contrast, increasing <i>Ter</i> copy number within the array reduced HR induced by the I-SceI homing endonuclease, but produced no consistent bias toward LTGC. Thus, the mechanisms governing HR at Tus/<i>Ter</i>-arrested replication forks are distinct from those governing HR at an enzyme-induced chromosomal double strand break (DSB). We propose that increased spatial separation of the 2 arrested forks encountering an extended Tus/<i>Ter</i> barrier impairs the coordination of DNA ends generated by the processing of the stalled forks, thereby favoring aberrant LTGC over conservative STGC.</p

Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/<i>Ter</i> mammalian replication fork barrier

<div><p>Classical non-homologous end joining (C-NHEJ) and homologous recombination (HR) compete to repair mammalian chromosomal double strand breaks (DSBs). However, C-NHEJ has no impact on HR induced by DNA nicking enzymes. In this case, the replication fork is thought to convert the DNA nick into a one-ended DSB, which lacks a readily available partner for C-NHEJ. Whether C-NHEJ competes with HR at a non-enzymatic mammalian replication fork barrier (RFB) remains unknown. We previously showed that conservative “short tract” gene conversion (STGC) induced by a chromosomal Tus/<i>Ter</i> RFB is a product of bidirectional replication fork stalling. This finding raises the possibility that Tus/<i>Ter</i>-induced STGC proceeds <i>via</i> a two-ended DSB intermediate. If so, Tus/<i>Ter</i>-induced STGC might be subject to competition by C-NHEJ. However, in contrast to the DSB response, where genetic ablation of C-NHEJ stimulates HR, we report here that Tus/<i>Ter</i>-induced HR is unaffected by deletion of either of two C-NHEJ genes, <i>Xrcc4</i> or <i>Ku70</i>. These results show that Tus/<i>Ter</i>-induced HR does not entail the formation of a two-ended DSB to which C-NHEJ has competitive access. We found no evidence that the alternative end-joining factor, DNA polymerase θ, competes with Tus/<i>Ter</i>-induced HR. We used chromatin-immunoprecipitation to compare Rad51 recruitment to a Tus/<i>Ter</i> RFB and to a neighboring site-specific DSB. Rad51 accumulation at Tus/<i>Ter</i> was more intense and more sustained than at a DSB. In contrast to the DSB response, Rad51 accumulation at Tus/<i>Ter</i> was restricted to within a few hundred base pairs of the RFB. Taken together, these findings suggest that the major DNA structures that bind Rad51 at a Tus/<i>Ter</i> RFB are not conventional DSBs. We propose that Rad51 acts as an “early responder” at stalled forks, binding single stranded daughter strand gaps on the arrested lagging strand, and that Rad51-mediated fork remodeling generates HR intermediates that are incapable of Ku binding and therefore invisible to the C-NHEJ machinery.</p></div

Loss of <i>Xrcc4</i> does not perturb HR regulation of Tus/<i>Ter</i>-induced STGC and LTGC.

<p><b>A</b>, Frequencies of Tus/<i>Ter</i>-induced repair <i>Xrcc4</i>^Δ/Δ clone 11 6x<i>Ter</i>-HR reporter clones stably transduced with pHIV-EV (lentiviral empty vector control) or with pHIV-<i>mXrcc4</i> (HA-tagged mouse Xrcc4 lentiviral expression vector) with selection of transduced cells in 100 μg/mL NTC. Cells were transiently co-transfected with empty or 3xMyc-NLS Tus expression vectors and siRNAs as shown. Each plot represents the mean of duplicate samples from eight independent experiments (n = 8). Error bars: s.e.m. <b><i>Xrcc4</i></b>^<b>Δ/Δ</b> <b>clone #11 pHIV-EV</b>: Tus-induced Total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0005; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC vs</i>. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0153; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.1481; si<i>LUC</i> vs. si<i>RAD51</i> p = 0.0034; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.2292; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0018. Tus-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0003; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. <b><i>Xrcc4</i></b>^Δ<b>/</b>Δ <b>clone #11 pHIV-<i>mXrcc4</i></b>: Tus-induced Total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0002; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001;si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0023; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0240; si<i>LUC</i> vs. si<i>RAD51</i> p = 0.0002; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.7398; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0022. Tus-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0004; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.0049; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0051. <b>B</b>, Frequencies of I-SceI-induced repair <i>Xrcc4</i>^Δ/Δ clone 11 6x<i>Ter</i>-HR reporter clones stably transduced with pHIV-EV (lentiviral empty vector control, “EV”) or with pHIV-<i>mXrcc4</i> (HA-tagged mouse Xrcc4 lentiviral expression vector, “X4”) with selection of transduced cells in 100 μg/ml NTC. Cells were co-transiently transfected with empty, or 3xMyc-NLS I-SceI expression vectors and siRNAs as shown. Each plot represents the mean of duplicate samples from eight independent experiments (n = 8). Error bars: s.e.m. <b><i>Xrcc4</i></b>^Δ<b>/</b>Δ <b>clone #11 pHIV-EV</b>: I-SceI-induced total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001 si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.3335; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0006. I-SceI-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0020; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.6260. <b><i>Xrcc4</i></b>^Δ<b>/</b>Δ <b>clone 11 pHIV-<i>mXrcc4</i></b>: I-SceI-induced total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0002; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.0590; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0001. I-SceI-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0011; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0330; si<i>LUC</i> vs. si<i>RAD51</i> p = 0.0491; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.0017; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0136. <b>C</b>, RT qPCR analysis of <i>BRCA1</i>, BRCA2, <i>CtIP</i> and <i>SLX4</i> mRNA in siRNA-treated <i>Xrcc4</i>^Δ<i>/</i>Δ cells stably transduced with pHIV-EV (“EV”) or pHIV-<i>mXrcc4</i> (“X4”) derived lentivirus. Data normalized to <i>GAPDH</i> and expressed as fold difference from si<i>LUC</i> sample from the same experiment (x = -2^ΔΔCt, with ΔΔCt = [Ct _target-Ct_Gapdh]-[Ct_si<i>LUC</i>-Ct_{si<i>GAPDH</i>}]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV_{<i>TARGET</i>}² + SDEV_<i>GAPDH</i>²]). <b>D</b>, Western blot of RAD51 protein abundance in siRNA-treated stably transduced <i>Xrcc4</i>^Δ<i>/</i>Δ cells; pHIV-empty vector control (“EV”) or pHIV-<i>mXrcc4</i> (“X4”). <b>E</b>, Western blot of Brca1 protein abundance in siRNA-treated stably transduced <i>Xrcc4</i>^Δ<i>/</i>Δ cells; pHIV-empty vector control (“EV”) or pHIV-<i>mXrcc4</i> (“X4”).</p

Impact of <i>Xrcc4</i> deletion on Tus/<i>Ter</i>-induced and I-SceI-induced HR.

<p><b>A</b>, Schematic of 6x<i>Ter</i>-HR reporter and HR repair products of Tus-<i>Ter</i>-induced fork stalling. Green box: wt<i>GFP</i>. Grey boxes: mutant <i>GFP</i>. Open ovals A and B: 5’ and 3’ artificial <i>RFP</i> exons. 5’Tr-<i>GFP</i>: 5’-truncated <i>GFP</i>. Orange triangle: 6x<i>Ter</i> element array. Navy blue line: I-SceI endonuclease cut site. STGC, LTGC: short tract and long tract gene conversion HR repair outcomes. LTGC generates wt<i>RFP</i> through RNA splicing (red filled ovals). <b>B</b>, <i>Xrcc4</i> gene structure in <i>Xrcc4</i>^fl/fl ES cells. <i>Xrcc4</i>^Δ<i>/</i>Δ allele lacks exon 3. Black triangles: <i>loxP</i> sites. Grey boxes: <i>Xrcc4</i> Exons 2–4. Location and direction of Exon3 genotyping primers a, a’, and b as indicated by arrows. Gel: PCR products for <i>Xrcc4</i>^fl/fl ES clones 8 and 39, and <i>Xrcc4</i>^Δ<i>/</i>Δ clones 11 and 13. <b>C</b>, RT qPCR analysis of <i>Xrcc4</i> expression in <i>Xrcc4</i>^fl/fl or <i>Xrcc4</i>^Δ<i>/</i>Δ clones. <i>Xrcc4</i> expression normalized to <i>GAPDH</i> and displayed as fold difference from <i>Xrcc4</i>^fl/fl clone 8 of the same experiment (x = -2^ΔΔCt, with ΔΔCt = [Ct_Xrcc4-Ct_Gapdh]-[Ct_<i>Xrcc4</i>-Ct_<i>GAPDH</i>]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV_<i>Xrcc4</i>² + SDEV_<i>GAPDH</i>²]). Xrcc4 abundance by Western blot in <i>Xrcc4</i>^fl/fl clones 8 and 39, and <i>Xrcc4</i>^Δ<i>/</i>Δ clones 11 and 13 cell protein extracts. <b>D</b>, Representative primary FACS data for two <i>Xrcc4</i>^fl/fl and two <i>Xrcc4</i>^Δ/Δ 6x<i>Ter</i>-HR reporter clones, as indicated, transfected with empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. FACS plots produced from pooled data of duplicate samples from three independent experiments. Numbers represent percentages. <b>E</b>, Frequencies of Tus/<i>Ter</i>-induced and I-SceI-induced repair in five independently derived <i>Xrcc4</i>^fl/fl (orange triangles, red squares) or <i>Xrcc4</i>^Δ/Δ (blue diamonds, navy blue circles) 6x<i>Ter</i>-HR reporter clones transiently transfected with empty, Tus or I-SceI expression vectors. Each dot plot represents the mean of duplicate samples from three independent experiments (n = 3), values are corrected for transfection efficiency–see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007486#sec008" target="_blank">Materials and Methods</a>. Error bars: standard error of the mean (s.e.m.). One-way ANOVA (Analysis of Variance) test comparing trend in HR between five <i>Xrcc4</i>^fl/fl and five <i>Xrcc4</i>^Δ/Δ clones: Tus-induced HR, total HR, p = 0.0017; STGC, p = 0.0015; LTGC, p = 0.7142; LTGC/(Total HR), p = 0.2636. I-SceI-induced HR, total HR, p<0.0001; STGC, p<0.0001; LTGC, p<0.0001; LTGC/(Total HR), p<0.0001. T-test comparing <i>Xrcc4</i>^fl/fl <i>vs</i>. <i>Xrcc4</i>^Δ/Δ clone pooled data, Tus-induced HR: total HR, p<0.0001; STGC, p<0.0001; LTGC, p = 0.6864; LTGC/(Total HR), p = 0.0332; I-SceI-induced HR, total HR, p<0.0001; STGC, p<0.0001; LTGC, p<0.0001; LTGC/(Total HR), p<0.0001.</p

Impact of <i>Ku70</i> deletion on Tus/<i>Ter</i>-induced HR.

<p><b>A</b>, <i>Ku70</i> mutant cell genotyping and gene structure in <i>Ku70</i>^–/–ES cells. <i>Ku70</i> null allele lacks Exon 4 and partially Exon 5. Grey boxes: <i>Ku70</i> Exons 3–6. Location and direction of Exons 4 and 5 and <i>neoR</i> genotyping primers a, a’, and b as indicated by arrows. Sa: SacI restriction site. Sc: ScaI restriction site. Gel: PCR products for single wild type control and nine <i>Ku70</i>^–/–ES 6x<i>Ter</i>-HR reporter clones (27, 34, 35, 38, 41, 45, 47, 54, and 92). <b>B</b>, Frequencies of Tus/<i>Ter</i>-induced and I-SceI-induced repair in nine independently derived <i>Ku70</i>^–/– 6x<i>Ter</i>-HR reporter clones (27, 34, 35, 38, 41, 45, 47, 54, and 92) transiently transfected with empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. Each dot plot represents the mean of duplicate samples from six independent experiments (n = 6), values are corrected for transfection efficiency. Error bars: s.e.m. One-way ANOVA (Analysis of Variance) test comparing trend in HR between nine <i>Ku70</i>^–/–clones: Tus-induced HR, total HR, p = 0.0205; STGC, p = 0.0173; LTGC, p = 0.1698; LTGC/(Total HR), p = 0.0261. I-SceI-induced HR, total HR, p = 0.0005; STGC, p = 0.0004; LTGC, p = 0.927; LTGC/(Total HR), p = 0.0081.</p

Distinct patterns of Rad51 recruitment to the Tus/<i>Ter</i> fork barrier and to a conventional DSB.

<p>A, Schematic of the 1x<i>GFP</i> 6x<i>Ter</i> reporter. Location of telomere (TEL) and centromere (CEN) shown. Red half-arrow heads: primer pairs. Grey box: mutant <i>GFP</i> allele <i>(</i>6x<i>Ter</i>-I-SceI-<i>GFP</i>). Orange triangle: 6x<i>Ter</i> element array. Navy blue line: <i>I-SceI</i> endonuclease target site and I-SceI site-specific guide RNA target site. Primer pair positions indicated as distance between the proximal end of the closer primer sequence to proximal edge of the 6x<i>Ter</i> array. B and C, Rad51 protein abundance at sites near the 6x<i>Ter-I-SceI-GFP</i> allele in response to Tus/<i>Ter</i>-induced replication fork stalling or DSB induction at 24 or 48 hours after transfection. Cells transiently transfected with empty vector (grey), pcDNA3β-myc NLS-Tus-F140A-3xHA (orange), pcDNA3β-myc NLS-I-SceI (royal blue), or co-transfected with spCas9 expression plasmid with control (white) or <i>I-SceI</i> site-specific (navy blue) guide RNA. Fold enrichment of Rad51 protein calculated as the mean 2^{-ΔΔ<i>CT</i>} from three independent experiments (n = 3) normalized against untreated controls (empty vector or guide RNA controls) and <i>β-Actin</i> control locus. Error bars indicate the standard deviation of the Δ<i>CT</i> measurement calculated as the change in Ct value obtained from the proximal-<i>Ter</i> locus and that obtained from <i>β-Actin</i> control locus.</p

Stable re-expression of wt<i>Xrcc4</i> does not affect Tus/<i>Ter</i>-induced HR in <i>Xrcc4</i>^Δ/Δ cells.

<p><b>A</b>, RT qPCR analysis of <i>Xrcc4</i> expression in stably transduced <i>Xrcc4</i>^fl/fl or <i>Xrcc4</i>^Δ<i>/</i>Δ clones. <i>Xrcc4</i> expression normalized to <i>GAPDH</i> and displayed as fold difference from <i>Xrcc4</i>^fl/fl parental reporter clone 8 of the same experiment (x = -2^ΔΔCt, with ΔΔCt = [Ct_Xrcc4-Ct_Gapdh]-[Ct_<i>Xrcc4</i>-Ct_<i>GAPDH</i>]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV_<i>Xrcc4</i>² + SDEV_<i>GAPDH</i>²]). <b>B</b>, Xrcc4 protein abundance by Western blot in extracts of parental <i>Xrcc4</i>^fl<i>/</i>fl clone #8 and <i>Xrcc4</i>^Δ<i>/</i>Δ clone #11 and derivative cultures stably transduced with empty lentiviral vector (pHIV-NAT-hCD52, “EV”) or HA-tagged mouse Xrcc4 lentiviral expression vector (“X4”). <b>C</b>, Fold enrichment of cultures transiently expressing exogenous GFP. Results represent fold enrichment of cultures transiently co-transfected with pcDNA3beta and <i>GFP</i>-expression plasmid co-cultured cells transiently transfected with pcDNA3beta alone. Each plot represents the mean of triplicate samples from three independent experiments (n = 3), fold enrichment GFP+ cells normalized to 0 μg/mL phleomycin control. Error bars: s.e.m. <b>D</b>, Frequencies of Tus/<i>Ter</i>-induced and I-SceI-induced repair in <i>Xrcc4</i>^fl/fl clone #8 or <i>Xrcc4</i>^Δ/Δ clone #11 6x<i>Ter</i>-HR reporter cells lentivirally transduced with pHIV-NAT-hCD52-EV (empty vector control) or pHIV-NAT-hCD52-<i>mXrcc4</i> (expressing HA-tagged mouse Xrcc4 expression vector) with selection of transduced cells in 100 μg/ml NTC. Cells were transiently transfected with empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. Each plot represents the mean of duplicate samples from six independent experiments (n = 6). Error bars: s.e.m. Tus-induced Total HR, t-test: flox8 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.0662; del11 +Xrcc4 <i>vs</i>. del11 +EV p = 0.4509; del11 +EV <i>vs</i>. flox8 +EV p = 0.6719; del11 +Xrcc4 <i>vs</i>. flox8 +Xrcc4 p = 0.0588; del11 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.5025. Tus-induced STGC, t-test: flox8 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.0836; del11 +Xrcc4 <i>vs</i>. del11 +EV p = 0.4126; del11 +EV <i>vs</i>. flox8 +EV p = 0.6144; del11 +Xrcc4 <i>vs</i>. flox8 +Xrcc4 p = 0.0595; del11 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.7215. Tus-induced LTGC, t-test: flox8 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.6686; del11 +Xrcc4 <i>vs</i>. del11 +EV p = 0.5972; del11 +EV <i>vs</i>. flox8 +EV p = 0.5313; del11 +Xrcc4 <i>vs</i>. flox8 +Xrcc4 p = 0.3007; del11 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.7870. Tus-induced LTGC/Total HR ratio, t-test: flox8 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.9182; del11 +Xrcc4 <i>vs</i>. del11 +EV p = 0.2133; del11 +EV <i>vs</i>. flox8 +EV p = 0.4686; del11 +Xrcc4 <i>vs</i>. flox8 +Xrcc4 p = 0.8360; del11 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.5771. I-SceI-induced Total HR, t-test: flox8 +Xrcc4 <i>vs</i>. flox8 +EV: p = 0.1292; del11 +Xrcc4 <i>vs</i>. del11 +EV p<0.0001; del11 +EV <i>vs</i>. flox8 +EV p<0.0001; del11 +Xrcc4 <i>vs</i>. flox8 +Xrcc4 p = 0.1030; del11 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.8690. I-SceI-induced STGC, t-test: flox8 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.1353; del11 +Xrcc4 <i>vs</i>. del11 +EV p<0.0001; del11 +EV <i>vs</i>. flox8 +EV p<0.0001; del11 +Xrcc4 <i>vs</i>. flox8 +Xrcc4 p = 0.0939; del11 +Xrcc4 <i>vs</i>. flox39 +EV p = 0.0081. I-SceI-induced LTGC, t-test: flox8 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.1840; del11 +Xrcc4 <i>vs</i>. del11 +EV p<0.0001; del13 +EV vs. flox39 +EV p<0.0001; del11 +Xrcc4 <i>vs</i>. flox8 +Xrcc4 p = 0.7589; del11 +Xrcc4 <i>vs</i>. flox39 +EV p = 0.1347. I-SceI-induced LTGC/Total HR ratio, t-test: flox8 +Xrcc4 <i>vs</i>. flox8 +EV p = 0.5908; del11 +Xrcc4 <i>vs</i>. del11 +EV p = 0.0001; del11 +EV <i>vs</i>. flox8 +EV p = 0.0001; del11 +Xrcc4 <i>vs</i>. flox8 +Xrcc4 p = 0.3729; del11 +Xrcc4 <i>vs</i>. flox39 +EV p = 0.4615.</p

Hypothetical models of Tus/<i>Ter</i>-induced HR.

<p><b>A,</b> Conventional DSB intermediate model. Dual incision of bidirectionally arrested forks generates DNA ends that are processed for HR. Unknown mechanisms prevent Ku access to the DNA ends at the stalled fork. Dark blue: parental strands. Light blue: nascent strands. Half arrows indicate direction of nascent strand synthesis. Orange triangles: Tus/<i>Ter</i> RFB. Green circles: Rad51 monomers. <b>B,</b> Template switch/fork reversal model. Rad51 is loaded onto exposed ssDNA lagging strand daughter strand gaps at the arrested fork. Following replisome disassembly, Rad51 mediates fork remodeling via a template switch mechanism. This process displaces the 3’ ssDNA end of the nascent leading strand, which is rapidly coated with RPA (not shown) followed by Rad51. The DNA end thus generated is incapable of binding Ku, excluding engagement of C-NHEJ. Further processing of the reversed fork may liberate the DNA end by more extensive fork reversal (not shown) and/or <i>via</i> incision of the 4-way reversed fork structure (red arrowhead). Although processing of the two opposing forks is depicted here as sequential, this model is also compatible with synchronous remodeling of both forks. Symbols as in panel A. Pale green circles, Rad51 monomers displaced from lagging strand.</p

Impact of CtIP depletion on repair frequencies in the presence or absence of hKU70.

<p><b>A</b>, Frequencies of Tus/<i>Ter</i>-induced repair in three independently derived <i>Ku70</i>^–/– 6x<i>Ter</i>-HR reporter clones (clones 27, 41, and 47) transiently expressing exogenous <i>hKU70</i> and transfected with siRNAs shown. Cells transiently co-transfected with empty pcDNA3beta or pcDNA3beta-<i>hKU70</i> expression vector and empty or 3xMyc-NLS Tus expression vectors treated with either si<i>LUC</i> or si<i>CtIP</i>. Each column represents the mean of duplicate samples from eight independent experiments (n = 8), values are corrected for transfection efficiency. Error bars: s.e.m. Tus-induced total HR, si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.0030; #41 +EV, p = 0.0207; #47 +EV, p = 0.0070; #27 +h<i>KU70</i>, p = 0.0047; #41 + h<i>KU70</i>, p = 0.0281; #47 + h<i>KU70</i>, p = 0.0148; Tus-induced STGC, t test: #27 +EV, p = 0.0011; #41 +EV, p = 0.0104; #47 +EV, p = 0.0070; #27 +h<i>KU70</i>, p = 0.0030; #41 + h<i>KU70</i>, p = 0.0207; #47 + h<i>KU70</i>, p = 0.0148; Tus-induced LTGC, t test: #27 +EV, p = 0.2786; #41 +EV, p = 0.5737; #47 +EV, p = 0.1304; #27 +h<i>KU70</i>, p = 0.8785; #41 + h<i>KU70</i>, p = 0.5737; #47 + h<i>KU70</i>, p = 0.5737; Tus-induced LTGC/(Total HR), t test: #27 +EV, p = 0.0002; #41 +EV, p = 0.0006; #47 +EV, p = 0.0006; #27 +h<i>KU70</i>, p = 0.0019; #41 + h<i>KU70</i>, p = 0.0030; #47 + h<i>KU70</i>, p = 0.0650; One-way ANOVA (Analysis of Variance) test comparing trend in Tus-induced Total HR: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p = 0.0002; si<i>LUC</i> all, p = 0.8904; si<i>CtIP</i> all, p = 0.1322. Tus-induced STGC, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p<0.0001; si<i>LUC</i> all, p = 0.9108; si<i>CtIP</i> all, p = 0.1155. Tus-induced LTGC, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p = 0.4334; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.3194; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p = 0.4144; si<i>LUC</i> all, p = 0.6254; si<i>CtIP</i> all, p = 0.2231. Tus-induced LTGC/(Total HR), one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.0004; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p = 0.0012; si<i>LUC</i> all, p = 0.9449; si<i>CtIP</i> all, p = 0.2989. <b>B</b>, Frequencies of I-SceI-induced repair in three independently derived <i>KU70</i>^Δ/Δ 6x<i>Ter</i>-HR reporter clones. Cells transiently co-transfected with empty pcDNA3beta or pcDNA3beta-<i>hKU70</i> expression vector and empty or 3xMyc-NLS I-SceI expression vectors treated with either si<i>LUC</i> or si<i>CtIP</i>. Each column represents the mean of duplicate samples from eight independent experiments (n = 8), values are corrected for transfection efficiency. Error bars: s.e.m. I-SceI-induced total HR, si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.0379; #41 +EV, p = 0.0281; #47 +EV, p = 0.0499; #27 +h<i>KU70</i>, p = 0.0003; #41 + h<i>KU70</i>, p = 0.0019; #47 + h<i>KU70</i>, p = 0.0011; I-SceI-induced STGC, si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.0379; #41 +EV, p = 0.0281; #47 +EV, p = 0.0379; #27 +h<i>KU70</i>, p = 0.0002; #41 + h<i>KU70</i>, p = 0.0019; #47 + h<i>KU70</i>, p = 0.0011; I-SceI-induced LTGC, si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.1104; #41 +EV, p = 0.7984; #47 +EV, p = 0.3282; #27 +h<i>KU70</i>, p = 0.3282; #41 + h<i>KU70</i>, p = 0.1949; #47 + h<i>KU70</i>, p = 0.1949; I-SceI-induced LTGC/(Total HR), si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.0011; #41 +EV, p = 0.0379; #47 +EV, p = 0.0070; #27 +h<i>KU70</i>, p = 0.0006; #41 + h<i>KU70</i>, p = 0.0650; #47 + h<i>KU70</i>, p = 0.0070; I-SceI-induced Total HR, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.0139; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p<0.0001; si<i>LUC</i> all, p<0.0001; si<i>CtIP</i> all, p<0.0001. I-SceI-induced STGC, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.0106; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p<0.0001; si<i>LUC</i> all, p<0.0001; si<i>CtIP</i> all, p<0.0001. I-SceI-induced LTGC, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.1503; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p = 0.1010; si<i>LUC</i> all, p = 0.0010; si<i>CtIP</i> all, p<0.0001. I-SceI-induced LTGC/(Total HR), one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p<0.0001; si<i>LUC</i> all, p = 0.0147; si<i>CtIP</i> all, p<0.0001. <b>C</b>, Observed repair frequencies for Tus or I-SceI induced HR, STGC and LTGC expressed as the ratio of si<i>CtIP</i> frequency/si<i>LUC</i> frequency for data from clones 27, 41 and 47 shown in panels <b>A</b> and <b>B</b>. One-way ANOVA test: Tus-induced total HR, p = 0.0779; Tus-induced STGC, p = 0.0564; Tus-induced LTGC, p = 0.2067. One-way ANOVA test: I-SceI-induced total HR, p<0.0001; I-SceI-induced STGC, p<0.0001; I-SceI-induced LTGC, p = 0.0832. <b>D</b>, RT qPCR analysis of <i>CtIP</i> mRNA in siRNA-transfected <i>Ku70</i>^–/–clones. Data normalized to <i>GAPDH</i> and expressed as fold difference from si<i>LUC</i> sample from the same experiment (x = -2^ΔΔCt, with ΔΔCt = [Ct_siCtIP-Ct_Gapdh]-[Ct_si<i>LUC</i>-Ct_{si<i>GAPDH</i>}]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV_<i>CtIP</i>² + SDEV_<i>GAPDH</i>²]).</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