Pdest frt t0 gfp brca1

Pyridostatin (PDS) was obtained from Cayman Chemical (#18013). TmPyP4 was from Calbiochem (#613560). Hoechst dye was from Santa Cruz Biotechnology (#sc-394039). Antibodies against synapsin were from Synaptic Systems (#106002). Antibodies against BRCA1 (#ab191042) and 53BP1 (#ab172580) were from Abcam. Antibodies against γH2A.X (JBW301) were from EMD Millipore (#05636). Rabbit antibodies against MAP2c (H-300, #sc-20172), and mouse antibodies against MAP2c (A-4, #sc-74421) were from Santa Cruz Biotechnology. Antibodies against β-actin (8H10D10) were from Cell Signaling (#3700). Anti-rabbit Alexa Fluor 488–labeled (#A11008), anti-mouse Alexa Fluor 488–labeled (#A11001), anti-rabbit Alexa Fluor 546–labeled (A11010), and anti-mouse Alexa Fluor 546-labeled (#A11003) secondary antibodies were from Life Technologies. pCAG-mApple-hTP53BP1 (1220-1709aa) was synthesized by Vectorbuilder. pSANG10-3F-BG4 was from Addgene (#55756; deposited by Dr. Shankar Balasubramanian, the University of Cambridge). pDEST-FRT/T0-GFP-BRCA1 was from Addgene (#71116; deposited by Dr. Durocher, the University of Toronto).

For live-cell studies, we used the following plasmids: GFP-tagged HP1β [42 (link)]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany). The plasmids were introduced into E. coli DH5α, and the DNA was isolated using the Qiagen Plasmid Maxi Kit (#121693; QIAGEN, Bio-Consult, Praha, Czech Republic). The cells were transfected with 2-5 μg plasmid DNA using METAFECTANE (#T020–1.0, Biontex Laboratories GmbH, München, Germany) [35 (link)].

BRCA1 gene was amplified and STOP codon removed from pDEST-FRT/T0-GFP-BRCA1 (Addgene #71116) by PCR using BP-tailed primers and cloned into pDNOR207 by Gateway® cloning BP reaction (Thermo Fisher Scientific). BRCA1-I26A mutation was introduced by site-directed mutagenesis. RAD18-pDNOR223 plasmid was obtained from Open Biosystems (Clone 1782) and the STOP codon was removed by site-directed mutagenesis. TULIP2 constructs were generated by Gateway® cloning LR reaction between donor plasmids containing BARD1^{28 (link)}, BRCA1 or RAD18 and acceptor TULIP2 plasmids^{27 (link)}. For the GFP-tagged constructs, the LR reactions were performed with pLVU-GFP, gift from Lars Ittner (Addgene #24177)^{59 (link)}. Primers are listed in Supplementary Table 1.