Branch Migrating Sister Chromatid Junctions Form at Replication Origins through Rad51/Rad52-Independent Mechanisms  Massimo Lopes, Cecilia Cotta-Ramusino, Giordano Liberi, Marco Foiani  Molecular Cell  Volume 12, Issue 6, Pages 1499-1510 (December 2003) DOI: 10.1016/S1097-2765(03)00473-8

Figure 1 Joint DNA Molecules Form at ARS305 Replication Origin ARS305 and A–D represent the restriction fragments analyzed; restriction sites are indicated as follows: EcoRV (E), HindIII (H), NcoI (N). W303-1A cells were presynchronized by α factor (αF) treatment in G1 and released into fresh YPD; DNA was prepared from cells collected at the indicated times using “CTAB extraction” (see Experimental Procedures), cut with EcoRV and HindIII, and analyzed by 2D gel, by sequential hybridization of the same membrane with the different probes. Molecular Cell 2003 12, 1499-1510DOI: (10.1016/S1097-2765(03)00473-8)

Figure 2 The Joint DNA Molecules Are Sensitive to the DNA Extraction Procedure and Able to Branch Migrate Samples were collected 45 min after the αF release using the conditions described in Figure 1. In (A), three samples were processed following different DNA extraction procedures (see Experimental Procedures); 2D gels were hybridized with the ARS305 probe. In (B), three DNA samples were extracted using the conditions described in Figure 1; DNA were digested with EcoRV and HindIII and subjected to electrophoresis in the first dimension; one of the agarose lanes was then processed for the second dimension under standard conditions (Control), while the other two were incubated in branch migration buffer at 65°C for 2 or 4 hr before running the second dimension. In (C), agarose slices from the first dimension gel were incubated prior to the second dimension in branch migration buffer, either in the presence or in the absence of 10 mM MgCl2 (see Experimental Procedures). Quantification of the signals is presented. Molecular Cell 2003 12, 1499-1510DOI: (10.1016/S1097-2765(03)00473-8)

Figure 3 Formation of Joint Molecules at ARS305 is RAD51- and RAD52 Independent rad51Δ (CY2269) and rad52Δ (CY2272) strains were released from G1 into fresh medium, under unperturbed conditions. DNA samples were processed as in Figure 1 and 2D gels hybridized to detect ARS305 and region A. Note that in these strains the activation of ARS305 is detectable 5–10 min earlier than in wt cells likely due to the presence of very large cells that need less time to reach the critical mass to enter S phase and therefore activate early origins of replication slightly in advance. The kinetics of formation and resolution of joint molecules is anticipated accordingly. Molecular Cell 2003 12, 1499-1510DOI: (10.1016/S1097-2765(03)00473-8)

Figure 4 Kinetics of DNA Replication and Joint Molecules Distribution in MMS- and HU-Treated Wild-Type Cells (A) W303-1A cells were presynchronized by αF treatment in G1 and released into fresh YPD containing 0.033% MMS. Samples were collected at the indicated times and processed as in Figure 1. (B) Presynchronized W303-1A cells were released in YPD containing 0.2 M HU. DNA was extracted as in Figure 1 from samples collected at the indicated times, digested with NcoI, and run in 2D gels. Membranes were hybridized with the ARS305 probe. Molecular Cell 2003 12, 1499-1510DOI: (10.1016/S1097-2765(03)00473-8)

Figure 5 Kinetics of DNA Replication and Joint Molecules Distribution in MMS- and HU-Treated rad53 Cells (A) rad53-K227A (CY2034) cells were presynchronized by αF treatment in G1 and released into fresh YPD containing 0.033% MMS. Samples were collected at the indicated times and processed as in Figure 1. (B) Presynchronized rad53-K227A cells were released in YPD containing 0.2 M HU. Samples were collected at the indicated times and processed as in Figure 4B. Molecular Cell 2003 12, 1499-1510DOI: (10.1016/S1097-2765(03)00473-8)

Figure 6 A Model Representing Hypothetical Hemicatenane Transitions under Physiological or Pathological Situations (A) A single-stranded junction between replicated fragments (hemicatenane) might form early after origin activation, and undergo local isomerization to form a pseudo double Holliday junction (by pairing of newly synthesized strands), or branch migration, eventually coiling together nonhomologous sequences. (B) Joint molecules degenerate in HU-treated rad53 cells. When junctions between newly replicated strands migrate and reach stalled forks deprived of DNA polymerases, a fraction of them might be resolved into single-stranded gaps, while others could form reversed forks (see Discussion for further details). Molecular Cell 2003 12, 1499-1510DOI: (10.1016/S1097-2765(03)00473-8)