Research Paper Volume 11, Issue 9 pp 2852—2873

Poly(ADP-ribosyl)ation and DNA repair synthesis in the extracts of naked mole rat, mouse, and human cells

Figure 1. Efficiency of PAR synthesis (A) and degradation (B) in WCEs. (A) PAR synthesis was performed for 1 min at 37 °C in the reaction mixture containing standard buffer components and 0.6 A260/mL activated DNA, 0.5 mg/mL cell extract proteins (or 10 nM recombinant human PARP1), and 20 μM [32P]NAD+. The reaction mixtures were treated and analyzed as described in the section ‘Synthesis and degradation of PAR in the extracts. PARP activity assay’. The yield of PAR analyzed by SDS-PAGE (the gel is shown in Fig. S1) is represented as a bar chart in arbitrary phosphorimager units. The analysis of PAR synthesis for three independent experiments is shown in numerical form under the bar chart. The data are the mean ± SD. In each experiment, the amount of PAR synthesized in the extract was normalized to that synthesized by 10 nM recombinant PARP1. (B) The reaction mixtures containing standard components, [32P]PAR synthesized as described in the section ‘Synthesis and degradation of PAR in the extracts. PARP activity assay’, and 0.5 mg/mL cell extract proteins or 10 nM recombinant PARG were incubated at 37 °C for different time intervals. Aliquots were further processed and analyzed as described in the section ‘Synthesis and degradation of PAR in the extracts. PAR degradation assay’. The amount of [32P]PAR in an equal aliquot of the control mixture (no proteins added) before incubation was taken as 100%. The points on the experimental curves represent the average of three independent experiments. Standard deviation did not exceed 10%.