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. 2013 Jun;33(12):2485–2496. doi: 10.1128/MCB.01406-12

Fig 4.

Fig 4

CAPNS1 depletion is linked to a defect in DNA repair. CAPNS1 (CS1)-depleted or LACZ-depleted U2OS cells were transfected with GFP7–pol-η and, where indicated, with wild-type USP1 (USP1 wt) or catalytically inactive USP1. Twenty hours later, the cells were irradiated with 30 J/m2 UV light or left untreated. Five hours later, the cells were fixed and analyzed by immunofluorescence. (a) Typical distribution of GFP–pol-η in control cells (left picture) and upon UV light-induced focus formation (right picture). (b) Percentage of GFP–pol-η focus-containing cells obtained in three independent experiments. (c) U2OS cells were transfected with LACZ, CAPNS1, or USP1 siRNA and subsequently with irradiated marker plasmid. The plasmid was recovered from the transfected cells 24 h after transfection and utilized to transform bacteria and measure repair efficiency in a standard supF mutagenesis assay. The graph reports the increase in mutagenesis occurring in CAPNS1- or USP1-depleted cells compared to control cells. (d) Wild-type, Capns1 KO, and Capns1-rescued (R) MEFs were irradiated with 30 J/m2 UV light or left untreated, and lysates were prepared 5 h later to monitor the PCNA ubiquitination (Ub-PCNA) level by Western blotting. (e) Wild-type and Capns1 KO MEFs were irradiated or not with 30 J/m2 UV light and utilized to perform a comet assay 48 and 65 h later; typical images are shown. (f) Quantification of parameters indicative of DNA damage and recovery: tail moment, % of DNA in the tail, and olive moment. CTRL, control. (g) CAPNS1 depletion is coupled to a decrease in USP1, and therefore the equilibrium between deubiquitinated and ubiquitinated PCNA is shifted toward the ubiquitinated form. Ubiquitinated PCNA recruits pol-η on DNA replication forks and may increase mutagenesis if it is not replaced by high-fidelity DNA polymerase once the lesion is bypassed. (h) Effect of CAPNS1 silencing on another USP1 substrate, ID2. Seventy-two hours after transfection with the indicated siRNAs, U2OS cells were lysed and analyzed with the indicated antibodies.