Journal: The EMBO Journal
Article Title: The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress
Figure Lengend Snippet: MMS22L–TONSL regulates RAD51 recruitment at stalled replication forks A iPOND assay schematic (top) and immunoblots (bottom) show effects of CPT and HU treatments on the RPA2 phosphorylation status in HeLa cells. Note that 30‐min treatment with 1 μM CPT stalls forks without inducing fork breakage, as the phosphorylation status of RPA2 using specific antibodies indicates the presence of pS33 associated with ssDNA rather than pS4/8 associated with DSBs (induced at later time point; 90 min). Asterisks indicate cross‐reactive bands. B Schematic representation (top) of TONSL, TONSL LRR*: E1089K and D1104N mutations within the LRR domain identified in ovarian cancer patient (COSMIC sample OCC06PT (Forbes et al )) and TONSL ΔLRR: C‐terminal TONSL truncation deleting the LRR domain; LRR, leucine‐rich repeats. Streptavidin pulldown (bottom) of indicated stably integrated HSS‐tagged‐TONSL variants overexpressed in HeLa cells. Input and coprecipitated proteins were detected by immunoblotting. ASF1A, known to interact with the N‐terminal region of TONSL (Duro et al ; O'Donnell et al ), was included as a control. C, D The presence of the indicated proteins at replication forks was analyzed by the iPOND‐CPT‐release assay in cells expressing either HSS‐tagged wild‐type TONSL, or TONSL LRR* or TONSL ΔLRR mutants and depleted for endogenous TONSL by siRNA targeting 3′UTR. The experiments were quantified and shown as average; n = 2 except for LRR* n = 1; error bars, SEM. TONSL and its interaction with MMS22L are required for normal levels of RAD51 at stalled replication forks.
Article Snippet: The following commercial antibodies were used: anti‐RAD51 (H‐92, Santa Cruz Biotech, 1:2,000), anti‐PCNA (PC10, Santa Cruz Biotech, 1:5,000), anti‐RPA2 (9H8, Imgenex, 1:2,000), anti‐RPA2 pS33 (A300–246A, Bethyl Laboratories, 1:1,000), anti‐RPA2 pS4/8 (A300–245A, Bethyl Laboratories, 1:1,000), anti‐MCM4 (clone B01P, Abnova, 1:1,000), anti‐α‐tubulin (DM1A, Sigma‐Aldrich, 1:10,000), anti‐HA.11 (16B12, Covance, 1:2,000), anti‐ASF1A (C6E10, Cell Signaling, 1:5,000), anti‐GAPDH (MAB374, Millipore, 1:5,000), and peroxidase‐conjugated anti‐mouse or anti‐rabbit antibodies (Pierce, 1:5,000).
Techniques: Western Blot, Cycling Probe Technology, Stable Transfection, Release Assay, Expressing