Expression constructs and site-directed mutagenesis

Human flag-tagged full-length TDP1 (FLAG-TDP1^WT), His-tagged and green fluorescent protein (GFP)-tagged TDP1 constructs were described previously (10,19). The FLAG-PRMT5 fusion construct was a kind gift from Dr Shilai Bao (Institute of Genetics and Developmental Biology, CAS, China). The flag-tagged N-terminal (1–293 aa) and C-terminal (294–637 aa) truncated PRMT5 and GFP-tagged N-terminal (1–185 aa) truncated TDP1 constructs were generated by polymerase chain reaction amplification using full-length PRMT5 or full-length TDP1 (FLAG-TDP1^WT) as template and were cloned in the mammalian expression vectors pCMV-Tag2 (Stratagene, La Jolla, CA, USA) or pEGFP-N2 vector (CLONTECH) respectively. The following point mutations: TDP1^R361K, TDP1^R586K, TDP1^{R361K, R586K} in FLAG and GFP tagged TDP1 constructs as well as His-TDP1^R361K,R586K were created using the ‘QuickChange’ protocol (Stratagene, La Jolla, CA, USA). All PCR-generated constructs were confirmed by DNA sequencing.