Break-induced replication repair of damaged forks induces genomic duplications in human cells

Abstract

In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers.

Fig. 1. POLD3 and POLD4 are required for cell cycle progression in the presence of oncogene-induced DNA replication stress

(A) Top, experimental outline. Bottom, flow cytometry analysis. U2OS cells expressed normal levels of cyclin E (NE) or overexpressed cyclin E (OE). E, EdU; B, BrdU; Noc, nocodazole; si, siRNA; EdU−/BrdU−, cells that remained in G1 or were blocked at the G1/S interface (G1); Edu−/BrdU+, cells that were in G1 at 0–1 h, but entered S phase by 7–8 h (G1->S); EdU+/BrdU+, cells that were in S phase at 0–1 h (S/G2). (B) Effect of POLD4 depletion on cell growth. The cells were seeded on Day 0; transfected with siRNA on Day 1 and counted on Day 4. Means and SDs from three independent experiments are shown.

Fig. 2. POLD3 and POLD4 are required for fork processivity under conditions of oncogene-induced DNA replication stress

(A) Distribution of replication forks. U2OS cells expressed normal levels of cyclin E (NE) or overexpressed cyclin E (OE). Replication forks were scored as ongoing (Ong), terminated (Term) or newly-fired (NewF). The data represent two independent experiments for POLD3 depletion and one experiment for POLD4 depletion. si, siRNA. (B) Distribution of replication speeds of ongoing DNA replication forks as a function of cyclin E expression levels and POLD3/POLD4 depletion. The percentages are relative to the total number of forks counted (ongoing, terminated and newly-fired), but only data for the ongoing forks are presented.

Fig. 3. Role of POLD3 and POLD4 in DNA DSB repair

(A) Percentages of cells with 53BP1 or RPA foci and average number of 53BP1 foci per cell in non-irradiated (0 Gy) and irradiated (2 Gy; 2, 16, 24 and 48 h after irradiation) U2OS cells. si, siRNA. (B) GFP-based reporter plasmid to monitor BIR. A beta-actin (βAct) promoter drives expression of N-terminal GFP sequences. The C-terminal GFP sequences are in the opposite orientation. The area of homology (Hom, 400 bps) is limited to one side of the I-SceI-induced break. Position 0 kb refers to the beta-actin promoter transcription start site. pA, poly A site. (C) Expected and observed products following repair by BIR. Three steps are shown: i) homology search; ii) strand extension; iii) dissolution of the D-loop and joining of the free DNA ends by MMEJ. P5GFP and P3GFP, primers to amplify GFP; P5EJ1, P5EJ2, P5EJ3 and P3EJ, primers to amplify MMEJ-generated junctions of types J1, J2 and J3, respectively. The short green/red lines indicate eight observed junctions. (D) Effect of POLD3 or POLD4 depletion on repair of DNA DSBs by BIR, SDSA and SSA. Means and SDs from experiments performed in quadriplicate are relative to control siRNA-transfected cells. si, siRNA.

Fig. 4. Cyclin E overexpression-induced genomic rearrangements

(A) Number of copy number alterations (CNAs) induced over a three week period in U2OS cells expressing normal or high levels of cyclin E (NE and OE, respectively). Means and SDs are from three clones per group. (B) Number of different types of CNAs (amplifications smaller than 200 kb, amplifications greater than 200 kb and deletions) induced in U2OS cells overexpressing cyclin E over a three week period, during which, the cells were transfected every three days with siRNA. The number of cell clones analysed is in parentheses. The data from the POLD3 and POLD4-depleted clones are also shown grouped together. si, siRNA. (C) Sequences of breakpoint junctions in two cyclin E-overexpressing clones, of which one had been transfected with control siRNA and one with siRNA targeting POLD3. The type of CNA associated with these junctions, the size of the CNA and the affected chromosome are indicated. The two joined sequences are colored blue and red, respectively; the microhomologies or small insertions at the junction are colored purple and brown, respectively.