RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress Rahul Bhowmick, Sheroy Minocherhomji, Ian D. Hickson  Molecular Cell  Volume 64, Issue 6, Pages 1117-1126 (December 2016) DOI: 10.1016/j.molcel.2016.10.037 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 64, 1117-1126DOI: (10.1016/j.molcel.2016.10.037) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 RAD52 Persists at Sites of On-Going DNA Synthesis in Mitosis Following Replication Stress (A) Previously proposed model for MiDAS. (B and C) Representative images (B) and quantification (C) of EdU incorporation (red) on isolated metaphase chromosomes (DAPI, blue) following replication stress (RS) (+APH). (D and E) Representative western blots of soluble and insoluble fractions (D) and quantification (E) following pre-treatment with (+APH) or without (−APH) APH. (F and G) Representative immunofluorescence (IF) images (F) and quantification (G) of the co-localization of RAD52 (green) with MUS81 or FANCD2 twin foci (red). DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 μm. See also Figure S1. Molecular Cell 2016 64, 1117-1126DOI: (10.1016/j.molcel.2016.10.037) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 RAD52 Is Essential for MiDAS and MUS81 Recruitment in Mitosis (A and B) Representative IF images (A) and quantification (B) of EdU foci (red) that co-localize with FANCD2 twin foci (green) in prometaphase cells following the indicated siRNA depletions (top panel) and RS. (C and D) Representative IF images (C) and quantification (D) of 53BP1 nuclear bodies (green) in G1 daughter cells following the indicated siRNA depletions (top panel) and RS. (E–G) Experimental workflow (E), representative IF images (F), and quantification (G) of EdU foci (red) after RAD52i. DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 μm. See also Figure S2. Molecular Cell 2016 64, 1117-1126DOI: (10.1016/j.molcel.2016.10.037) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 RAD52 Is Required for the Recruitment of MUS81 and POLD3 to Chromatin in Mitosis (A and B) Representative IF images (A) and quantification (B) of co-localized MUS81 foci (red) and FANCD2 twin foci (green). (C and D) Representative images (C) and quantification (D) of co-localized SLX4 foci (red) and FANCD2 twin foci (green). (E and F) Representative western blots (E) and quantification (F) of soluble and insoluble fractions after the indicated siRNA treatments (top). DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 μm. See also Figure S3. Molecular Cell 2016 64, 1117-1126DOI: (10.1016/j.molcel.2016.10.037) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Consequences of Defective MiDAS (A and B) Representative images (A) and quantification (B) of PICH-positive UFBs (red) with FANCD2 foci (green) at their bridge termini. (C–E) Experimental workflow (C), representative images (D), and quantification (E) of MiDAS (EdU foci, red) as indicated. DNA was stained using DAPI (blue). Data are means of three independent experiments. Error bars represent SEM. Scale bars, 10 μm. (F and G) Clonogenic assays (F) and quantification (G) of U2OS cells following indicated treatments in S-phase or mitosis. (H) Model proposing that MiDAS can occur via an MMBIR-like process. Replication fork disruption, perhaps due to the presence of DNA secondary structures at AT-rich regions or R-loops, followed by limited 5′ end resection of the generated DNA end, exposes a region of microhomology that can anneal with the partially single-stranded template DNA. The annealing step might require a DNA helicase to facilitate template denaturation. Processing of the resulting replication intermediate by the activated SLX-MUS complex (SLX4 in complex with MUS81-EME1 and other nucleases) in early mitosis is then associated with POLD3-dependent conservative DNA repair synthesis. This process would account for the high level of CNVs that arise at CFS loci in cancer cells. For clarity, the replication fork merging with the MiDAS bubble from the right is omitted. However, if this fork were also disrupted, DNA synthesis could arise on both sister chromatids via the same mechanism. See also Figure S4. Molecular Cell 2016 64, 1117-1126DOI: (10.1016/j.molcel.2016.10.037) Copyright © 2016 Elsevier Inc. Terms and Conditions