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. 2014 Oct 2;10(10):e1004654. doi: 10.1371/journal.pgen.1004654

Figure 3. Complementation of the polymerase activity of POLQ rescues DNA damage hypersensitivity in cells lacking Polq.

Figure 3

(A) POLQ cDNA was cloned into the pCDH-FH vector containing a FLAG-HA epitope tag on the c-terminus. 293T cells were transiently transfected with pCDH containing either empty-vector control or POLQ cDNA. (B) Crude extracts were immunoblotted with the indicated antibodies to confirm full-length expression of recombinant POLQ or (C) mutant constructs. (D) Stable MEF lines complemented with POLQ expression vectors (or empty vector control) were assayed for Polq expression by qPCR. WT4 and WT6 are independent clones complemented by wild-type POLQ; POL, mutation in the DNA polymerase domain; HLD, mutation in the DNA helicase domain, DM, mutation in both domains. (E) The complemented MEF lines were treated with bleomycin for 24 hr and cellular ATP levels were measured 72 hr later. (F) Spontaneous micronuclei and (G) DNA double-strand breaks (>2 colocalized γH2AX and 53BP1 foci per cell), quantified for three independent experiments. The brightness of the entire microscope field was increased to better display the fluorescence for publication, using Adobe Photoshop CS6.