We engineered a system in which ful

We engineered a system in which full-length Mre11 cDNA is stably
expressed from an integrated plasmid vector in cells that were Mre11cond/−. Endogenous Mre11 was subsequently removed through Cre-mediated conversion of Mre11cond/− to Mre11−/−, and independent
clones with Mre11 levels similar to endogenous were pursued for further study (Supplementary Fig. 3). Mre11 expressed from cDNA successfully reconstituted levels of Rad50 and NBS1 (Fig. 1c) and supported the ability of MRN to activate the ATM kinase, as determined by relative levels of ATM autophosphorylation (Fig. 1d)15,35. Unexpectedly, despite the apparent restoration of MRN, CtIP protein levels were not restored (Fig. 1c). This suggests that the mechanisms by which Mre11 influences CtIP levels as compared to RAD50 or NBS1 levels are distinct.
The expression system fused 54 amino acids to the Mre11
C terminus (hereafter termed Mre11C54), originating from three tags (His-V5-His). To test the possibility that this large addition prevents CtIP restoration, we reconstructed the plasmid to express Mre11 fused only to a short histidine tag composed of five amino acids (Mre11C5). Indeed, Mre11C5 expression restored CtIP (Fig. 1c). Thus, we hypothesized that the C terminus of Mre11 possesses an unknown function that controls CtIP levels. We therefore examined the impact of Mre11ATLD1 C-terminal deletion. Existing ATLD1 cell lines cannot be used to study specific roles of the Mre11 C terminus, as they also harbor low levels of the entire MRN complex30,31. To circumvent this limitation, we stably expressed Mre11ATLD1 from cDNA in Mre11−/− cells (Fig. 1c). When expressed to approximately endogenous levels, Mre11ATLD1 showed a pattern identical to the impact of Mre11C54 and restored NBS1 and Rad50
levels and ATM activation without restoration of CtIP (Fig. 1c,d). Thus, the C terminus of Mre11 possesses a distinct function required to maintain normal CtIP levels.

結果 (繁體中文) 1:

我們設計的全長 Mre11 cDNA 在穩定系統表示從綜合質粒載體中的儲存格被 Mre11cond / −。 內源性 Mre11 隨後被刪除通過介導 Cre 轉為 −，Mre11−/Mre11cond/− 和獨立與類似的內源性 Mre11 水準克隆被追求為進一步研究 (補充圖 3)。Mre11 表示從 cDNA 成功重組水準的 Rad50 和 NBS1 (圖 1 c) 並支援 MRN 能夠啟動 ATM 激酶，由 ATM 自動 (圖 1 d) 磷酸化的相對水準 15 歲，35 歲。出乎意料的是，儘管 MRN 明顯恢復，CtIP 蛋白水準未恢復 (圖 1 c)。這表明，其中 Mre11 影響 CtIP 水準與 RAD50 或 NBS1 級別的機制不同。表達系統融合到 Mre11 54 氨基酸C terminus (hereafter termed Mre11C54), originating from three tags (His-V5-His). To test the possibility that this large addition prevents CtIP restoration, we reconstructed the plasmid to express Mre11 fused only to a short histidine tag composed of five amino acids (Mre11C5). Indeed, Mre11C5 expression restored CtIP (Fig. 1c). Thus, we hypothesized that the C terminus of Mre11 possesses an unknown function that controls CtIP levels. We therefore examined the impact of Mre11ATLD1 C-terminal deletion. Existing ATLD1 cell lines cannot be used to study specific roles of the Mre11 C terminus, as they also harbor low levels of the entire MRN complex30,31. To circumvent this limitation, we stably expressed Mre11ATLD1 from cDNA in Mre11−/− cells (Fig. 1c). When expressed to approximately endogenous levels, Mre11ATLD1 showed a pattern identical to the impact of Mre11C54 and restored NBS1 and Rad50levels and ATM activation without restoration of CtIP (Fig. 1c,d). Thus, the C terminus of Mre11 possesses a distinct function required to maintain normal CtIP levels.

結果 (繁體中文) 3:

我們設計了一個系統，全長cDNA穩定
MRE11表示從一個集成的質粒載體，mre11cond /−細胞。內源性MRE11隨後被删除通過Cre介導的mre11cond /−轉換Mre11−/−，獨立
尅隆Mre11水准與內源性追求深造（補充圖3）。MRE11表達成功再造Rad50 NBS1基因水准（圖1c）和支持MRN啟動ATM激酶的能力，如ATM磷酸化水准相對確定（圖1d）約。出乎意料的是，儘管MRN明顯恢復，CtIP蛋白水准沒有恢復（圖1C）。這表明，Mre11影響CtIP水准相比Rad50或NBS1層面是不同的機制。
表達系統融合54個胺基酸的C末端MRE11
（以下簡稱mre11c54），來自三個標籤（his-v5-his）。測試這個大的加入封锁CtIP恢復的可能性，我們重建了質粒表達MRE11融合只有一個由五個胺基酸組成的短的組氨酸標籤（mre11c5）。事實上，mre11c5表達恢復CTIP（圖1c）。囙此，我們推測，C端具有MRE11未知函數控制CtIP水准。囙此我們研究mre11atld1 C端缺失的影響。現有的atld1細胞不能用於研究MRE11 C末端的特定角色，他們心懷低水準整個MRN complex30,31。為了繞過這個限制，我們穩定表達從發生−/−細胞cDNA mre11atld1（圖1C）。當表示為約內生水准，mre11atld1模式顯示相同的影響和恢復mre11c54 NBS1蛋白
水准和ATM啟動沒有恢復的CTIP（圖1C、D）。囙此，Mre11基因的C末端具有一個獨特的功能維持正常水准所需的資訊平臺。