Pcmv6 an 3ddk

The plasmid-expressing 3xFlag-tagged human Pur-α cDNA (NCBI accession number: NM_008989) was generated by subcloning Pur-α cDNA into the vector pCMV6-AN-3DDK (OriGene). The plasmid encoding Myc-tagged mouse DUSP8 was generated by cloning the Dusp8 cDNA into the vector pMTSM-Myc (6 (link)). The plasmid expressing DUSP8 (C246S) phosphatase–dead mutant was generated from the Myc-DUSP8 plasmid by mutating cysteine-246 to serine using PCR-based site-directed mutagenesis. The plasmids expressing YFP-fused DUSP8 and CFP-fused Pur-α proteins were constructed by subcloning the Dusp8 cDNA and Pur-α cDNA from the Myc-Dusp8 plasmid and 3xFlag-Pur-α plasmid into the pCMV6-AC-Yfp vector (OriGene) and pCMV6-AN-Cfp vector (OriGene), respectively. The plasmid expressing GFP-fused Pur-α protein was generated by subcloning the human Pur-α cDNA into the vector pCMV6-AN-Gfp (OriGene). The plasmids encoding Pur-α (S217A) and Pur-α (T183A) mutant proteins were generated by mutating the indicated amino acid residues in the 3xFlag-Pur-α or GFP-Pur-α plasmid.

The mammalian expression vector containing the hSSB1 CDS (pCMV6-AN-3DDK) was supplied by Origene. Site-directed mutagenesis (SDM) was used to introduce the non-coding mutations for small interfering RNA (siRNA) resistance and was performed using the polymerase Pfu Ultra (Stratagene). The pET28a hSSB1 vector has been described previously. For the preparation of truncation mutants, premature STOP codons were introduced by SDM as per above. The preparation of hOGG1 point mutants has been performed on pGEX-hOGG1 vector, as described earlier. Primer sequences are listed in Supplementary Table S1.
Mammalian expression vectors were transfected using Lipofectamine 2000 (Life Technologies).
Stealth siRNA against hSSB1 were synthesized by Life technologies (Invitrogen). Individual siRNA sequences were (sense) 5′-GACAAAGGACGGGCAUGAGdTdT and (antisense) 5′-CUCAUGCCCGUCCUUUGUCdTdT (17 (link)). hOGG1 was targeted using either pooled esiRNAs (Sigma Aldrich) or the Silencer Select siRNA sequences (sense) 5′-GAUCAAGUAUGGACACUGAtt and (antisense) 5′-UCAGUGUCCAUACUUGAUCcg (Life Technologies). siRNAs were transfected using RNAiMax (Life Technologies).