Fig. 2: PARylated Polθ is inhibited in DNA binding and TMEJ.
From: PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks
a EMSA showing that PARylated Polθ-hel is inhibited in ssDNA binding. Cy3-ssDNA probe and PARP1 activating substrate (PAS) are indicated. b Denaturing gel showing that the presence of PARP1 and NAD+ inhibit Polθ-hel ATPase activity (left).% inorganic phosphate (PI) formation indicated. pssDNA substrate indicated. Bar plot showing mean ± SD results from ATPase assay (right). ****p < 0.0001 and ***p < 0.001 using unpaired two-tailed t test, statistics from three biological replicates (1 vs 3 p = 0.001119, 2 vs 3 p = 0.000025, 3 vs 4 p = 0.000045). c EMSA showing that the presence of PARP1 and NAD+ inhibit PolθΔcen ssDNA binding. Cy3-ssDNA probe and PARP1 activating substrate (PAS) are indicated. d Schematic of in vitro TMEJ assay using pssDNA substrates containing 3′ terminal microhomology. e Denaturing gel showing XmaI cleavage of expected TMEJ product. Schematic of pssDNA substrate and TMEJ synapse with XmaI recognition site indicated in red (top). f Denaturing gel showing that the presence of PARP1 and NAD+ suppress Polθ-pol and PolθΔcen TMEJ activities. g Bar plots showing mean% TMEJ activity ± SD by Polθ-pol and PolθΔcen in the presence of the indicated factors. ****p < 0.0001 using unpaired two-tailed t-test, statistics from three biological replicates (1 vs 3 p = 0.00002, 2 vs 3 p = 0.00001, 3 vs 4 p = 0.000025, 5 vs 7 p = 0.000086, 6 vs 7 p = 0.000011, 7 vs 8 p = 0.000035). h Denaturing gel showing that the presence of PARP1 and NAD+ suppress FL-Polθ TMEJ activity. i Bar plots showing mean ± SD results from%TMEJ activity by FL-Polθ and in the presence of the indicated factors. ****p < 0.0001 using unpaired two-tailed t-test, statistics from three biological replicates (1 vs 3 p = 0.000048, 2 vs 3 p < 0.00001, 3 vs 4 p < 0.00001). j Denaturing gel showing that olaparib prevents suppression of Polθ-pol TMEJ in the presence of PARP1 and NAD+. Experiments a, c, e, j were repeated at least two times with similar results. Source data are provided as a Source Data file.
