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. 2019 Oct 24;47(22):11709–11728. doi: 10.1093/nar/gkz912

Figure 7.

Figure 7.

The APE1 DNA incision and POLβ nucleotide incorporation activities are also defective in the lamin A/C siRNA-depleted U2OS cells; moreover, the BER defect in these cells is PARylation dependent. (A) APE1 DNA incision product generation from extracts of siScr and siLamin A/C U2OS cells (representative image); incision activity was determined as the intensity of product band relative to the combined intensities of substrate and product bands (n = 3, mean ± SD). (B) POLβ nucleotide incorporation product formation from extracts of siScr and siLamin A/C U2OS cells (representative image); incorporation activity was determined as the combined intensity of the three incorporation bands (one, two and three nucleotide incorporation) relative to the combined intensity of the three incorporation bands plus the intensity of the substrate (i.e. ‘unrepaired’) band (n = 3, mean ± SD). (C) FPG-comet assay. DNA repair efficiency was expressed as percent of FPG-sensitive sites remaining after 8 h of repair, relative to 5 min of repair, after correction for the FPG-sensitive sites in untreated cells (n = 100 comet tails, mean ± SEM). Cell culture medium was supplemented with NAD+ (2 mM) or NAD+ together with PARP1 inhibitor 3-AB (10 mM), as indicated in the figure, for 2 h prior to H2O2 treatment and sustained for the duration of the repair period. (D) Western blot of triplicate lysates from Lmna+/+ MEFs transfected with either siScr or siLamin A/C siRNA. (E) Model for the role of lamin A/C in BER. New pathway insight from this study is shown in the gray shaded box. Loss of lamin A/C leads to reduction in APE1 DNA incision and POLβ nucleotide incorporation activities that negatively affect the rate of BER. This leads to accumulation of oxidized bases. Chronic exposure to oxidative stress leads to enhanced DNA base substitution mutations in lamin A/C-deficient cells that are signatures of oxidative damage persistence. As previously characterized, lamin A/C depletion also leads to defective double strand break (DSB) repair. Like the BER defect, this can lead to genomic instability and contribute to aging and cancer. †In Lmna−/− MEFs (but not in siLamin A/C U2OS cells) there was reduced expression of PARP1, LIG3 and POLβ; ††The other main two DNA lesion types that accumulate when BER rate is impaired are alkylated and deaminated bases; †††Tested in MEFs (but not in U2OS cells); Pos, positive control (purified APE1 or POLβ instead of lysate), Neg, negative control (no lysate). P values were determined by Student's t-test; ***P < 0.0001; **P < 0.005; *P < 0.05.