We excluded a role for RAP1 in inhibiting RNF168 recruitment as deletion of the RAP1-interaction motif (amino acids 286–299)17 in the context of the TRFcT construct did not result in 53BP1 localization to telomeres (Fig. 3a, b and Supplementary Fig. 11b). Similarly, deletion of the TIN2-interaction motif (amino acids 352–367)18 resulted only in a minor induction of 53BP1 accumulation, thus excluding a critical role for this interaction in the suppression of RNF168 at telomeres (Fig. 3a, b and Supplementary Fig. 11b). This implicated the C-terminal portion of the hinge domain (amino acids 407–431), a region that by sequence alignment shows a high degree of conservation between species (Supplementary Fig. 10). Deletion of this region—which we have termed the inhibitor of DDR (iDDR) region—in the context of the TRFcT construct resulted in levels of 53BP1 that are comparable to those observed in Trf2−/− cells (Fig. 3a, b and Supplementary Fig. 11). To further validate this finding and to test whether this region is also sufficient to prevent DDR activation, we expressed the iDDR region in the context of TRF1 (Fig. 3a). Notably, the resulting TRF1iDDR protein can complement the phenotypes associated with TRF2 loss, with significant inhibition of 53BP1 and RNF168 localization and chromosome fusions (Fig. 3b, c and Supplementary Figs 11b, f and 12).
译文
我们排除一个角色在抑制RNF168 RAP1招聘为删除的RAP1-interaction主题(氨基酸286 - 299)17日在上下文的TRFcT构造并未导致53 bp1定位到染色体端粒(图3 a,b和补充无花果。11 b)。同样,删除的TIN2-interaction主题(氨基酸352 - 367)18结果只有一个小感应53 bp1积累,因此不包括一个关键的角色在这个交互抑制RNF168在端粒(图3 a,b和补充无花果。11 b)。这暗示,c末端部分的铰链域(氨基酸407 - 431),一个地区,通过序列比对显示了一个高程度的保护物种之间(补充图10)。删除这个裁决我们称为抑制剂的DDR(iDDR)地区的背景下TRFcT构造导致53 bp1的水平,可以观察到在Trf2−−/细胞(图3 a,b和补充无花果。11)。为了进一步验证这一发现和测试这个地区是否也足以防止DDR激活,我们表示iDDR地区上下文中的TRF1(图3)。值得注意的是,由此产生的TRF1iDDR蛋白质可以补充相关的表型TRF2损失,显著抑制53 bp1和RNF168本地化和染色体融合(图3 b、c和补充无花果11 b,f和12)。
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