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. 2016 Jun 9;7(34):54430–54444. doi: 10.18632/oncotarget.9914

Figure 4. SAF-A phosphorylation by DNA-PK is linked to transient dissociation of NEIL1 from chromatin.

Figure 4

A-B. Enhanced pS59 SAF-A formation in the HEK293/ U2OS cell chromatin after irradiation. Chromatin-bound NEIL1 was released at ~15-30 min after IR exposure and restored after 1h, shown in quantitative histogram (lower panel, for HEK293). C. Chromatin-bound NEIL1 level was not altered by pretreatment with the DNA-PKcs inhibitor NU7441, which prevented SAF-A phosphorylation. NU7441 (10 μM) pretreatment for 2 h followed by exposure to 3 Gy X-rays and chromatin extraction from cells harvested at the indicated times. NEIL1's transient dissociation from chromatin coincided with increase in the 53BP1 level, consistent with NHEJ repair of overt DSBs, while BER was delayed. Quantitative histograms shown in lower panels. D. WT SAF-A, but not the non-phosphorylatable S59A mutant, negatively regulated chromatin-bound NEIL1. HEK293 cells were transfected with 3′ UTR hnRNP-U siRNA and FLAG-hnRNP-U WT or S59A, followed by irradiation. E. PLA analysis revealed increased interaction of SAF-A(S59A), but not the WT or S59D mutant with NEIL1 after irradiation. HEK293 cells were transfected with FLAG-tagged WT, S59A, or S59D SAF-A plasmid, and 48 h later, were irradiated (3 Gy). The PLA was performed for NEIL1 and FLAG -SAF-A after further incubation for 30 min.