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. 2015 Nov 16;112(48):E6624–E6633. doi: 10.1073/pnas.1508543112

Fig. S8.

Fig. S8.

Dysregulation of DNA elongation after UV irradiation is not dependent on the exonuclease activity of Mre11. (A) Extent of Mre11 down-regulation. U2OS cells treated with two different siRNAs for Mre11 (Mre11-1 corresponds to sequence 1, which was used in the experiments described in the main text, and Mre11-2 corresponds to sequence 2) were subjected to RT-PCR analysis. (B) U2OS cells transfected with the indicated siRNAs were UV-irradiated and subjected to the fiber-labeling CldU20IdU20 protocol. CldU track length was quantified. (C) IdU track lengths for the experiment in B were quantified. (D) U2OS cells were transfected with control or Rad51-1 siRNAs and treated with mirin for 30 min before UV irradiation and throughout the end of the experiment. IdU track length distribution at 20 min after UV irradiation was calculated. (E) IdU track length distribution at 60 min after UV irradiation (20 J/m2). Samples that were not treated with mirin are shown as circles, and samples treated with mirin are represented by triangles. (F) Speculative model depicting the substrates for DNA degradation in RAD51-deficient samples. In Rad51-depleted samples, UV-triggered degradation of nascent strands depends on the exonuclease activity of Mre11. In agreement with previous work by others (29), our data support the notion that the degradation takes place behind the fork at ssDNA gaps formed during DNA replication in the absence of exogenous damage. (G) Extent of Primpol down-regulation by siRNA. Two siRNAs were used [Primpol-1(sequence 1), used in the experiments described in the main text and Primpol-2 (sequence 2) used in H and I]. Samples were subjected to RT-PCR. (H) U2OS cells transfected with the indicated siRNAs were sham-irradiated, and IdU length distribution at 60 min was calculated. (I) IdU length distribution at 60 min after UV irradiation. (J) Western blot analysis performed 24 h after cotransfection of GFP-polκ plasmid and control or polκ siRNAs. Antibodies specific for GFP and Ku70 were used. (K) IdU track length distribution at 60 min in untreated samples transfected with control, Rad51, polη, Primpol, and/or polκ siRNAs as indicated. (L) IdU track length distribution at 60 min after UV (20 J/m2) irradiation in samples transfected with control, Rad51, polη, Primpol, and/or polκ siRNAs as indicated.