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BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks

Abstract

Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases1,2,3. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes4,5,6,7,8. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks9. Although BRCA1 and BRCA2 affect replication fork processing10,11,12, direct evidence that BRCA gene products regulate homologous recombination at stalled chromosomal replication forks is lacking, due to a dearth of tools for studying this process. Here we report that the Escherichia coli Tus/Ter complex13,14,15,16 can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mouse cells. Tus/Ter-induced homologous recombination entails processing of bidirectionally arrested forks. We find that the Brca1 carboxy (C)-terminal tandem BRCT repeat and regions of Brca1 encoded by exon 11—two Brca1 elements implicated in tumour suppression—control Tus/Ter-induced homologous recombination. Inactivation of either Brca1 or Brca2 increases the absolute frequency of ‘long-tract’ gene conversions at Tus/Ter-stalled forks, an outcome not observed in response to a site-specific endonuclease-mediated chromosomal double-strand break. Therefore, homologous recombination at stalled forks is regulated differently from homologous recombination at double-strand breaks arising independently of a replication fork. We propose that aberrant long-tract homologous recombination at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells.

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Figure 1: Tus/Ter-induced replication fork stalling in mammalian cells.
Figure 2: Tus/Ter-induced homologous recombination in mammalian cells.
Figure 3: The Brca1 tandem BRCT repeat regulates Tus/Ter-induced homologous recombination.
Figure 4: Brca1 Exon11 regulates Tus/Ter-induced homologous recombination.

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Acknowledgements

We thank B. Michel for reagents and advice; D. Livingston, K. Marians, J. Walter, I. Hickson, S. Powell, V. Zakian and members of the Scully laboratory for helpful discussions; I. Hickson and S. Powell for sharing their unpublished data on Tus/Ter replication blocks in eukaryotes; R. Baer for antibodies and A. Ashworth for Brca1 conditional ES cell line 11CO/47T. This work was supported by NIH grants R01CA095175, R01GM073894 and R21CA144017 (to R.S.). N.A.W. was supported by an NIH/NCI postdoctoral fellowship (5T32CA081156) and an ACS postdoctoral research fellowship (PF-12-248-01-DMC). C.F. was supported by NIH grant R37GM26938 (to V.A.Z.).

Author information

Authors and Affiliations

Authors

Contributions

N.A.W., G.C. and R.S. designed experiments. N.A.W., G.C., B.H. and A.K. performed experiments. C.F. provided expert advice on execution of two-dimensional gel electrophoresis experiments. C.D. generated Brca1 exon 11 conditional ES cells. N.A.W. analysed the data. N.A.W. and R.S wrote the manuscript.

Corresponding author

Correspondence to Ralph Scully.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Tus/Ter-induced replication fork stalling visualized by additional restriction digests.

a, Phosphorimager quantification of spot B. One of five independent experiments that contributed to Fig. 1c. Four areas were quantified for each sample using ImageJ 1.48p software, as shown by cartoon. A, area containing a portion of replication fork arc A. B, area containing replication fork stall spot B (same shape/size as A). F, largest area of replication fork arc that is accessible to quantification in every sample. G, same shape as F, used to quantify background signal on membrane. Stall spot B intensity was calculated as: (B−A)/(F−G)×100%. Note, this value does not correspond to the probability of stalling at the Tus/Ter block, but is used to illustrate the relatively weaker arrest produced by 6×REVTer. b, Plasmid elements as in Fig. 1a. MluI/XmnI digested plasmid yields a linear fragment of 5.4 kb. Probe for Southern blotting indicated by the black bar. c, Plasmid replication intermediates extracted from 293E cells transiently transfected with 6×Ter-containing plasmids or no Ter control, co-transfected with empty vector (EV), TusH144A or Tus as shown. All samples are from one experiment. Plasmid DNA extracted from 293E cells was digested with XmnI and MluI and analysed by neutral/neutral two-dimensional gel-electrophoresis and Southern blotting. Replication intermediates as described in Fig. 1a. d, Predicted replication intermediates generated by Tus/Ter-induced replication fork stalling with or without effective FR/EBNA1 replication fork block. Diagrams below plasmid maps show shape of the major Tus/Ter-dependent fork arrest species. Green dotted line shows predicted additional branch of double Y structure formed by stalling of anticlockwise fork at Tus/6×Ter when FR/EBNA1 replication block fails. The length of the additional branch is shown in each diagram. Note that the relationship between spots B and C will vary according to the length of this additional branch. e, Plasmid replication intermediates extracted from 293E cells transiently transfected with 6×Ter-containing plasmids and co-transfected with empty vector (EV) or wild-type Tus as shown. Restriction digests of extracted plasmids as shown. All samples are from one experiment. Note: replication fork size and position of stall spot B in relation to replication arc A varies with restriction digest. For example, spot B in KpnI/MluI is close to the 2n linear position, since the Tus/Ter-stall site is only 680 bp from the KpnI site. For the same reason, spots B and C are closely placed in the KpnI/MluI-digested sample. Note: the relatively weak spot C in the KpnI/MluI digest, which is consistent across multiple experiments, might reflect a proportionately large contribution of ssDNA (reflecting processed lagging strand DNA22) to the 680 bp lagging strand of the stalled anticlockwise fork.

Extended Data Figure 2 Estimation of efficiencies of the FR/EBNA1 and Tus/6×Ter replication fork barriers.

a, Tus/Ter-mediated replication stall structures responsible for spots B and C. The relative abundance of the single stall spot B and the double Y stall spot C can be used to calculate the efficiency of the FR/EBNA1 replication fork barrier. b, Phosphorimager analysis of twelve independent Southern blot experiments (method described in Fig. 1b). Areas B, B’, C and C’ are the same shape and size within each experiment, but vary between experiments. B, stall spot B. B’, background gel signal of same area as B. C, stall spot C. C’, background gel signal of same area as C. Relative intensity of spot B/(B + C) estimates the stalling efficiency at FR/EBNA1 and is calculated as: (B − B’)/(B + C − B’ − C’)×100%. The stalling efficiency at FR/EBNA1 is therefore 70 ± 2.2% (s.e.m.). Relative intensity of spot C is calculated as: (C−C’)/(B + C − B’ −C’)×100%. c, Structure of p6×Ter-2Ori plasmid. Stalled replication intermediates depict different combinations of FR/EBNA1 block/bypass and Tus/6×Ter block/bypass. Spots B and B2 are defined as in the diagram. Spots C and C2 result from FR/EBNA1 bypass. Spot C2 requires successful arrest at both of the 6×Ter arrays. Spot C results from bypass of one of the two 6×Ter arrays. d, One of three independent experiments performed with p6×Ter-2Ori. Methods as in Fig. 1b. Note presence of four stall spots in p6×Ter-2Ori replicating in presence of Tus. Double Y stall spots C and C2 and background signal C’ were quantified. Note that the shape and size of each area is identical within an individual experiment, but varies between experiments. By considering only double Y stall spots (that is, in which FR/EBNA1 bypass has occurred), the relative abundance of the double Y stall spots C and C2 are used to estimate the efficiency of the Tus/6×Ter replication fork barrier. Let a = probability of the 6×Ter array blocking the fork and b = probability of 6×Ter bypass. Then a + b = 1. The probability of the two 6×Ter arrays blocking each fork on one p6×Ter-2Ori plasmid (generating spot C2) is a2. The probability of one 6×Ter array being blocked and the second array being bypassed (generating spot C) is 2ab. Relative densitometry of spots C and C2 (each with subtraction of background C’) shows that spot C contributes 49.6% and C2 contributes 50.4% (s.e.m. 5.6%). Therefore 0.496a2 = 0.504 × 2ab. Solving this, a = 0.67 Therefore the estimated efficiency of the Tus/6×Ter replication fork block within the replicating plasmid is 67%. Note that the efficiency of the Tus/6×Ter replication fork block within the chromosome is unknown.

Extended Data Figure 3 Two-ended versus one-ended break repair models of Tus/Ter-induced homologous recombination.

a, Bidirectional fork arrest would provide two DNA ends for sister chromatid recombination. Termination by annealing generates STGC products of a fixed size. Recombining GFP elements and HR reporter features other than Tus/Ter are not shown. Black strands represent parental DNA. Grey strands represent newly synthesized DNA. Arrowheads on DNA strands represent DNA synthesis. Blue/grey hexagons, Tus monomers. Red triangles, Ter sites. Green line, invading DNA strand. Green dotted line, nascent strand extension. b, Unidirectional fork arrest would provide only one DNA end for sister chromatid recombination. Following one-ended invasion of the neighbouring sister chromatid, any STGC products could not be terminated by annealing, as there is no homologous second end. Termination by non-canonical mechanisms would generate STGCs of unpredictable/variable size, as in ref. 21. DNA and protein elements labelled as in panel a. LTGC is not considered in this analysis, as the mechanisms of termination of the major LTGC products are not accessible from the current data. Each model invokes a hypothetical DSB intermediate. Tus/Ter-induced HR could be initiated by a template switching mechanism (that is, without the formation of an initiating DSB intermediate). However, the requirement for a homologous second end is not altered by consideration of a template switch model and this second end must be provided by the processing of a second arrested fork (the right-hand fork in panel a).

Extended Data Figure 4 Tus/Ter-induced homologous recombination in Brca1fl/BRCTTer/HR cells conforms to an affinity/avidity model.

a, Primary data from Fig. 2c, showing directly measured frequencies of background HR, Tus-induced HR and I-SceI-induced HR in three independent Brca1fl/BRCTTer/HR reporter clones. Cells were transfected with empty vector (EV, grey squares), myc-NLS-I-SceI (I-SceI, blue diamonds), or myc-NLS-Tus expression vectors (Tus, orange circles). Each point represents the mean of triplicate samples from three independent experiments (that is, n = 3). Error bars represent s.e.m. Student’s t-test of Tus versus EV: STGC P < 0.0001; LTGC P < 0.0001. Student’s t-test of I-SceI versus EV: STGC P < 0.0001; LTGC P < 0.0001. Student’s t-test of Tus versus I-SceI: STGC P < 0.0001; LTGC P = 0.0018; LTGC/Total HR P = 0.0186. b, Primary data comparing a single ROSA26 targeted Brca1fl/BRCTTer/HR clone with three independently derived clones, each harbouring a single intact 6×Ter/HR reporter randomly integrated at an unknown locus. Filled symbols, ROSA26-targeted clone (as in panel a). Open symbols, data from randomly integrated 6×Ter/HR reporter clones. Each point represents the mean of six independent experiments, triplicate replicates for each experiment (that is, n = 6). Error bars represent s.e.m. Student’s t-test of pooled random integrants Tus versus EV: STGC P < 0.0001; LTGC P < 0.0001. Student’s t-test of pooled random integrants I-SceI versus EV: STGC P < 0.0001; LTGC P < 0.0001. Student’s t-test of pooled random integrants Tus versus I-SceI: STGC P < 0.0001; LTGC P = 0.3620; LTGC/total HR P = 0.00012. c, Primary data of STGC products observed in Brca1fl/BRCTTer/HR cells transfected with empty vector (EV), wild-type Tus, DNA binding defective TusH144A, lock defective TusF140A or I-SceI. All expression vectors are codon-optimized for mammalian expression and encode N-terminal myc epitope and NLS sequences. Each column represents the mean of six independent experiments (that is, n = 6). Error bars represent s.e.m. Student’s t-test of Tus versus EV: P = 0.0002; Tus versus TusH144A: P = 0.0004; Tus versus TusF140A: P = 0.0042; Tus versus I-SceI: P = 0.0139; TusH144A versus EV: P = 0.4406; TusF140A versus EV: P < 0.0001; TusF140A versus TusH144A: P < 0.0001; TusF140A versus I-SceI: P = 0.0888. d, Myc-tagged protein abundance in transfected Brca1fl/BRCTTer-HR cells. EV, empty vector. Other lanes as marked. Lower panel, β-tubulin loading control. e, Cartoons of the Ter/HR reporter constructs assayed in panel f. f, Frequencies of Tus-induced STGC in Brca1fl/BRCT cells carrying single copy ROSA26-targeted Ter/HR reporters shown in panel e. Left, HR in 6×Ter, 3×Ter, 2×Ter and 1×Ter HR reporters, as shown. Right, HR in three independently derived clones carrying single copy, ROSA26-targeted 6×REVTer HR reporters. Each column represents the mean of three independent experiments (that is, n = 3). Error bars represent s.e.m. Student’s t-test of 6×Ter versus 3×Ter#1: P = 0.2604; 6×Ter versus 3×Ter#2: P = 0.5192; 6×Ter versus 2×Ter#1: P = 0.0547; 6×Ter versus 2×Ter#2: P = 0.0524; 6×Ter versus 1×Ter#1: P = 0.0507; 6×Ter versus 1×Ter#2: P = 0.0507; 3×Ter#1 versus 3×Ter#2: P = 0.8291; 3×Ter#1 versus 2×Ter#1: P = 0.0650; 3×Ter#1 versus 2×Ter#2: P = 0.0606; 3×Ter#1 versus 1×Ter#1: P = 0.0576; 3×Ter#1 versus 1×Ter#2: P = 0.0574; 3×Ter#2 versus 2×Ter#1: P = 0.1832; 3×Ter#2 versus 2×Ter#2: P = 0.1748; 3×Ter#2 versus 1×Ter#1: P = 0.1677; 3×Ter#2 versus 1×Ter#2: P = 0.1697. By one-way ANOVA (analysis of variance) test used to compare more than three sets of data, the trend in HR from 6× to 1×, P = 0.0012.

Extended Data Figure 5 Slx4/FancP depletion suppresses Tus/Ter-induced HR.

a, Frequencies of STGC in Brca1fl/BRCTTer-HR cells co-transfected with Tus (orange) or I-SceI (blue) and with either control Luciferase siRNA (siLuc), Slx4 SMARTpool (siSlx4), Slx1 SMARTpool (siSlx1), Slx1 and Slx4 SMARTpools (siSlx1 siSlx4), Eme1 SMARTpool (siEme1), Eme1 and Slx4 SMARTpools (siEme1 siSlx4), Xpf SMARTpool (siXpf), Xpf and Slx4 SMARTpools (siXpf siSlx4). Each column represents the mean of triplicate samples from four independent experiments for each clone (that is, n = 4). Error bars represent s.e.m. Tus-induced HR: Student’s t-test of siSlx4 versus siLuc: P = 0.0219; siSlx4 versus siSlx1: P = 0.0012; siSlx4 versus siSlx4 + Slx1: P = 0.5983; siSlx4 versus siEme1: P = 0.0171; siSlx4 versus siSlx4 + siEme1: P = 0.8721; siSlx4 versus siXpf: P = 0.0098; siSlx4 versus siSlx4 + siXpf: P = 0.4711; siSlx1 versus siLuc: P = 0.9332; siEme1 versus siLuc: P = 0.4631; siXpf versus siLuc: P = 0.7818; siSlx4 + siSlx1 versus siLuc: P = 0.0155; siSlx4 + siEme1 versus siLuc: P = 0.0215; siSlx4 + siXpf versus siLuc: P = 0.0305. I-SceI-induced HR: Student’s t-test of siSlx4 versus siLuc: P = 0.0907; siSlx4 versus siSlx1: P = 0.0195; siSlx4 versus siSlx4 + siSlx1: P = 0.4897; siSlx4 versus siEme1: P = 0.0568; siSlx4 versus siSlx4 + siEme1: P = 0.3411; siSlx4 versus siXpf: P = 0.0745; siSlx4 versus siSlx4 + siXpf: P = 0.2726; siSlx1 versus siLuc: P = 0.9198; siEme1 versus siLuc: P = 0.3349; siXpf versus siLuc: P = 0.9217; siSlx4 + siSlx1 versus siLuc: P = 0.1521; siSlx4 + siEme1 versus siLuc: P = 0.2864; siSlx4 + siXpf versus siLuc: P = 0.2063. b, qRT–PCR analysis of mRNA exon boundaries for Slx4, Slx1, Eme1 and Xpf mRNA in siRNA-SMARTpool-treated cells used in panel a.

Extended Data Figure 6 Southern blot analysis of Tus/Ter- and I-SceI-induced HR products in Brca1Δ/BRCTTer/HR cells.

a, Structure of the 6×Ter/HR parental reporter, and major STGC or LTGC HR products (assuming two-ended breaks). Elements as shown in Fig. 2a. b, Southern blot analysis of Tus-induced and I-SceI induced HR products in Brca1Δ/BRCTTer-HR cells. P, un-rearranged reporter; STGC and LTGC as shown. SN, STGC accompanied non-disjunction with retention of parental donor reporter; LN, LTGC accompanied non-disjunction with retention of parental donor reporter. B, BglII digest. BI, BglII + I-SceI digest. Membranes probed with full-length GFP cDNA. Panels underneath two SN events and one LN event show that re-cloning does not separate the two reporters, confirming that the cell contains two copies of the reporter (consistent with non-disjunction).

Extended Data Figure 7 Brca1 contributes quantitatively and qualitatively to homologous recombination at stalled replication forks.

a, Frequencies of Tus-induced and I-SceI-induced HR in Brca1fl/BRCT and Brca1Δ/BRCTTer/HR cells transiently co-transfected with Tus or I-SceI and with either control Luciferase siRNA (siLuc) or Brca1 SMARTpool (siBrca1). Each column represents the mean of triplicate samples for each independent clone from seven independent experiments (that is, n = 7). Error bars represent s.e.m. Tus-induced HR, Brca1fl/BRCT cells, Student’s t-test siBrca1 versus siLuc: STGC: P = 0.0013; LTGC: P = 0.0206; LTGC/total HR: P = 0.0003; Brca1Δ/BRCT cells, siBrca1 versus siLuc: STGC: P = 0.0016; LTGC: P = 0.4558; LTGC/total HR: P < 0.0001. I-SceI-induced HR, Brca1fl/BRCT cells, Student’s t-test siBrca1 versus siLuc: STGC: P < 0.0001; LTGC: P = 0.0033; LTGC/total HR: P = 0.9214; Brca1Δ/BRCT cells, siBrca1 versus siLuc: STGC: P = 0.0013; LTGC: P = 0.2348; LTGC/total HR: P = 0.0071. b, Brca1 protein levels and β-actin loading control in Brca1fl/BRCT and Brca1Δ/BRCT in siRNA-treated cells as shown. c, qRT–PCR analysis of Brca1 mRNA in siRNA-treated cells as shown.

Extended Data Figure 8 Brca2 contributes quantitatively and qualitatively to homologous recombination at stalled replication forks.

a, Frequencies of Tus-induced and I-SceI-induced HR in Brca1fl/BRCT and Brca1Δ/BRCTTer/HR cells transiently co-transfected with Tus, or I-SceI and with either control Luciferase siRNA (siLuc) or Brca2 SMARTpool (siBrca2). Each column represents the mean of triplicate samples for each independent clone from five independent experiments (that is, n = 5). Error bars represent s.e.m. Tus-induced HR, Brca1fl/BRCT cells, Student’s t-test siBrca2 versus siLuc: STGC: P = 0.0031; LTGC: P = 0.0007; LTGC/total HR: P = 0.0042; Brca1Δ/BRCT cells, siBrca2 versus siLuc: STGC: P = 0.0040; LTGC: P = 0.0013; LTGC/total HR: P = 0.0006. I-SceI-induced HR, Brca1fl/BRCT cells, Student’s t-test siBrca2 versus siLuc: STGC: P = 0.0028; LTGC: P = 0.0456; LTGC/total HR: P = 0.7945; Brca1Δ/BRCT cells, siBrca2 versus siLuc: STGC: P = 0.0010; LTGC: P = 0.2926; LTGC/total HR: P = 0.0316. b, qRT–PCR analysis of Brca2 mRNA in siRNA-treated cells as shown.

Extended Data Figure 9 Rad51 contributes quantitatively and qualitatively to homologous recombination at stalled replication forks.

a, Frequencies of Tus-induced and I-SceI-induced HR in Brca1fl/BRCT and Brca1Δ/BRCTTer/HR cells transiently co-transfected with Tus, or I-SceI and with either control Luciferase siRNA (siLuc) or Rad51 SMARTpool (siRad51). Each column represents the mean of triplicate samples for each independent clone from seven independent experiments for Brca1fl/BRCT (that is, n = 7) and four independent experiments for Brca1Δ/BRCT cells (that is, n = 4). Error bars represent s.e.m. Tus-induced HR, Brca1fl/BRCT cells, Student’s t-test siRad51 versus siLuc: STGC: P < 0.0001; LTGC: P = 0.1578; LTGC/total HR: P = 0.0002; Brca1Δ/BRCT cells, siRad51 versus siLuc: STGC: P = 0.0010; LTGC: P = 0.0676; LTGC/total HR: P < 0.0001. I-SceI-induced HR, Brca1fl/BRCT cells, Student’s t-test siRad51 versus siLuc: STGC: P = 0.0014; LTGC: P = 0.0002; LTGC/total HR: P = 0.6216; Brca1Δ/BRCT cells, siRad51 versus siLuc: STGC: P = 0.0068; LTGC: P = 0.2064; LTGC/total HR: P = 0.0186. b, Rad51 protein levels and β-tubulin loading control in Brca1fl/BRCT and Brca1Δ/BRCT siRNA-treated cells as shown.

Extended Data Figure 10 Effect of 53BP1 inhibition on Tus/Ter-induced homologous recombination.

a, Frequencies of Tus-induced and I-SceI-induced HR in Brca1fl/BRCT and Brca1Δ/BRCTTer/HR cells transiently co-transfected with Tus or I-SceI expression vectors and with either F53BP1 D1521R fragment (D1521R; non-chromatin-binding negative control for ‘dominant-negative’ 53BP1 fragment) or ‘dominant-negative’ F53BP1wt fragment (F53BP1wt). Each column represents the mean of triplicate samples for each independent clone from five independent experiments (that is, n = 5). Error bars represent s.e.m. Tus-induced HR, Brca1fl/BRCT cells, Student’s t-test D1521R versus F53BP1wt: STGC: P = 0.1818; LTGC: P = 0.9005; LTGC/total HR: P = 0.3570; Brca1Δ/BRCT cells, Student’s t-test D1521R versus F53BP1wt: STGC: P = 0.5008; LTGC: P = 0.5375; LTGC/total HR: P = 0.4921. I-SceI-induced HR, Brca1fl/BRCT cells, Student’s t-test D1521R versus F53BP1wt: STGC: P = 0.0442; LTGC: P = 0.5739; LTGC/total HR: P = 0.2250; Brca1Δ/BRCT cells, Student’s t-test D1521R versus F53BP1wt: STGC: P = 0.0086; LTGC: P = 0.6888; LTGC/total HR: P = 0.0328. Tus-induced LTGC/total HR, Brca1fl/BRCT versus Brca1Δ/BRCT cells, Student’s t-test F53BP1wt: 0.0064; Brca1fl/BRCT versus Brca1Δ/BRCT cells, Student’s t-test D1521R: 0.0014; I-SceI-induced LTGC/total HR, Brca1fl/BRCT versus Brca1Δ/BRCT cells, Student’s t-test F53BP1wt: 0.1556; Brca1fl/BRCT versus Brca1Δ/BRCT cells, Student’s t-test D1521R: 0.0208. b, Abundance of 53BP1 fragments, and β-tubulin (loading control) in treated Brca1fl/BRCT and Brca1Δ/BRCTTer/HR reporter ES cells in a.

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Willis, N., Chandramouly, G., Huang, B. et al. BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. Nature 510, 556–559 (2014). https://doi.org/10.1038/nature13295

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