( A) DNA synthesis in the absence of NAD +. Standard deviation did not exceed 10%.ĭNA synthesis and effect of PAR synthesis in WCEs. The points on the experimental curves represent the average of three independent experiments. The amount of PAR in an equal aliquot of the control mixture (no proteins added) before incubation was taken as 100%. Aliquots were further processed and analyzed 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 ☌ for different time intervals. ( B) The reaction mixtures containing standard components, PAR synthesized as described in the section ‘Synthesis and degradation of PAR in the extracts. In each experiment, the amount of PAR synthesized in the extract was normalized to that synthesized by 10 nM recombinant PARP1. The analysis of PAR synthesis for three independent experiments is shown in numerical form under the bar chart. S1) is represented as a bar chart in arbitrary phosphorimager units. The yield of PAR analyzed by SDS-PAGE (the gel is shown in Fig. The reaction mixtures were treated and analyzed as described in the section ‘Synthesis and degradation of PAR in the extracts. ( A) PAR synthesis was performed for 1 min at 37 ☌ in the reaction mixture containing standard buffer components and 0.6 A 260/mL activated DNA, 0.5 mg/mL cell extract proteins (or 10 nM recombinant human PARP1), and 20 μM NAD +. Our results suggest that MARylation/PARylation of DNA in the extracts depends on the ratios between PARPs and can be controlled by DNA-binding proteins.ĭNA polymerase Heterocephalus glaber Mus musculus base excision repair poly(ADP‐ribose) polymerases.Įfficiency of PAR synthesis (A) and degradation (B) in WCEs. PARP1/PARP2 can then transfer the ADP-ribose moieties onto initial ADP-ribose. The results obtained with WCEs, recombinant proteins and recently found ability of PARPs to attach the ADP-ribose moieties to DNA allowed us to attribute these products to primer mono(ADP-ribosyl)ation (MARylation) at the 5'-terminal phosphate by PARP3 during the DNA synthesis. Under conditions of PAR synthesis, the efficiency of DNA synthesis was only slightly enhanced in all extracts and in mouse WCEs unusual products of the primer elongation were detected. The level of PAR synthesis was more than ten-fold higher in human WCE as compared to rodent WCEs, while the efficiency of DNA synthesis was comparable. Here, using the whole-cell extracts (WCEs) of Hgl, mouse and human cells, we studied the interrelation between DNA synthesis on the substrates of base excision repair and the activity of poly(ADP-ribose) polymerases (PARPs) responsible for the transfer of the ADP-ribose moieties onto different targets. DNA repair capacity in cells of naked mole rat (Hgl), a species known for its longevity and resistance to cancer, is still poorly characterized.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |