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. 2019 Oct 14;17(10):e3000468. doi: 10.1371/journal.pbio.3000468

Fig 7. Phosphorylation of T156 and T145 sites in KHDC3L by ATM is required for HR repair and PARP1 activation.

Fig 7

(A) T156A, T145A, and Δ11 mutations caused similar level of defect in recruiting RAD51 to DNA DSB sites, whereas T156D had no effects on RAD51 recruitment. Double mutations of T156A/T145A displayed a more severe phenotype than those of individual mutation (n = 50 in one replicate, total three independent experiments). (B) PARP1 activity in hESCs with WT or mutant KHDC3L proteins. Neutral comet assay (C) (n = 200 from two independent experiments) and immunoblotting (D) confirmed that hESCs expressing Δ11 or T156A mutant KHDC3L accumulated a higher level of DNA DSBs than did WT hESCs or ESCs expressing T156D. (E) Phosphorylation of T145/T156 sites at different hESC lines. (F) T145A, T156A, and Δ11 mutations displayed similar level of defects in DSB repair (n = 200 from two independent experiments). Similar to KHDC3L−/−, T156A/T145A double mutations caused more-severe defects. (G) Complete repression of ATM activation by KU-55933 eliminated the phosphorylation of T145/T156 in KHDC3L, whereas inhibition of ATR activity by VE-821 had no effects. Student two-tailed t test was performed for statistical analysis. Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Underlying numerical values in (A), (C), and (F) can be found in S1 Data. Δ11, p.E150_V160del; ATM, Ataxia-telangiectasia mutated; ATR, Ataxia-telangiectasia and Rad3-related protein; DSB, double-strand break; ESC, embryonic stem cell; Etop, etoposide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hESC, human ESC; HR, homologous recombination; KHDC3L, KH domain containing 3 like; ns, not significant; PAR, poly(ADP-ribose); PARP, PAR polymerase; RAD51, RAS associated with diabetes protein 51; WT, wild type.