Increased Recombination Intermediates and Homologous Integration Hot Spots at DNA Replication Origins  Mónica Segurado, Marı́a Gómez, Francisco Antequera  Molecular Cell  Volume 10, Issue 4, Pages 907-916 (October 2002) DOI: 10.1016/S1097-2765(02)00684-6

Figure 1 Recombination Intermediates at ORI Regions in S. pombe Twelve intergenic genomic regions were analyzed by two-dimensional gel electrophoresis. As shown in the top row, replication bubbles (rb) and recombination intermediates (ri) (black arrows) were detected simultaneously at ORI-containing regions. Neither of them was detectable at passively replicated regions (R1–R6) (bottom row). All twelve intergenic regions analyzed contained promoters except R6, where the restriction fragment tested was located between two convergent transcription units. Molecular Cell 2002 10, 907-916DOI: (10.1016/S1097-2765(02)00684-6)

Figure 2 Resolution of Recombination Intermediates into Linear Fragments under Conditions that Promote Spontaneous Branch Migration S. pombe DNA was digested with BamHI, and three separated samples of the resulting fragments were subjected to the first dimension of the electrophoresis. One of the gel slices containing the restriction fragments was processed under standard conditions (Control), and the other two slices were incubated in branch migration buffer at 65°C for 3 or 4 hr before running the second dimension. After blotting, the membranes were successively hybridized to probes specific for ORI 19 and ORI 22. Numbers in the panels indicate the percentage of radioactivity in recombination intermediates before (Control) and after spontaneous branch migration. Recombination intermediates resolved into linear fragments are indicated by arrows. The proportion of bubble (B) and fork (F) intermediates in each case is indicated below the panels. Molecular Cell 2002 10, 907-916DOI: (10.1016/S1097-2765(02)00684-6)

Figure 3 Recombination Intermediates Parallel ORI Activity during S Phase S. pombe cdc10-129 mutant cells were arrested in G1 by incubation at the restrictive temperature. Upon release at 25°C, samples were taken at the indicated times during S phase, and the activation timing of seven genomic ORIs was monitored by two-dimensional gel electrophoresis. Bubble arcs and recombination intermediates are indicated by arrows. Recombination intermediates were detected only when ORIs were active, and differences in the time of firing of specific ORIs were paralleled by the appearance of recombination intermediates. Molecular Cell 2002 10, 907-916DOI: (10.1016/S1097-2765(02)00684-6)

Figure 4 ORI Firing Precedes the Appearance of Recombination Intermediates (A) S. pombe cdc10-129 mutant cells were arrested in G1 at 36°C for 4 hr and then released at 25°C in 10 mM hydroxyurea. Progression through S phase was monitored by FACS analysis. (B) Samples from the synchronized culture were taken at the indicated times, and the replication profile of ars1, ORI 17, and R4 was monitored. The same three filters were successively hybridized to probes specific for each of the three regions. Replication bubbles precede the appearance of recombination intermediates in the 1.5 hr sample, and these still persist after both replication origins are no longer active in the 3 hr (ars1) and 7 hr (ORI17) samples. Only passive replication arcs are detected at R4. Molecular Cell 2002 10, 907-916DOI: (10.1016/S1097-2765(02)00684-6)

Figure 5 Recombination Proteins Are Required for Efficient ORI Activity (A) Activation of ars1 and ORI 17 in asynchronous, exponentially growing S. pombe wild-type cells (wt) and in rad22Δ, rhp51Δ, and rhp54Δ mutants. The spike of recombination intermediates was not detectable in any of the mutants. Bubble arcs were significantly reduced at ars1 in rad22Δ and at both ORIs in rhp54Δ cells. They fell below the level of detection in rhp51Δ and at ORI 17 in rad22Δ. Aberrant intermediates in rad22Δ and rhp54Δ cells are indicated by open arrowheads. (B) Microscopy analysis and DAPI staining of wild-type cells and of rad22Δ, rhp51Δ, and rhp54Δ mutants shows that many cells in the population have aberrant morphology with deformed and fragmented nuclei. (C) FACS analysis indicates a wide heterogeneity in cell size and DNA content in the three mutants relative to wild-type cells. Molecular Cell 2002 10, 907-916DOI: (10.1016/S1097-2765(02)00684-6)

Figure 6 High Frequency of Integration at the ars1 Region Requires ORI Activity (A) Diagram of the ars1 intergenic region between the hus5+ gene and a tRNASer gene. Transcription and replication initiation sites are indicated (TIP and RIP). The ura4+ gene marker was targeted at the eight positions indicated by black arrows and numbered relative to RIP. (B) Colonies of S. pombe ura4-d18 per microgram of DNA capable of growing in minimal medium upon targeting the ura4+ gene marker at the eight sites indicated in (A). Data shown are averages of four independent experiments. Segments indicate standard deviation. (C) S. pombe cdc10-129 mutant cells were arrested at 36°C for 4 hr and then released at 25°C. Progression through S phase at the indicated times was monitored by FACS analysis. (D) Transformation efficiency upon targeting the ura4+ gene marker at position +39 in ars1 at the indicated times during a synchronous S phase as shown in (C). Data are averages of three independent experiments. The transformation efficiency is different in (B) and (D) because cells were transformed by the lithium acetate protocol and by electroporation, respectively. (E) Transformation efficiency upon targeting the ura4+ gene marker at the R5 region at the same time points and under identical conditions as in (D). The scale of transformants per microgram is 40-fold larger than in (D). Molecular Cell 2002 10, 907-916DOI: (10.1016/S1097-2765(02)00684-6)