Persistent 3'-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3'-phosphatase.
Persistent 3'-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3'-phosphatase.
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DOI：
10.1016/j.dnarep.2018.05.002
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发表时间：
2018-08
期刊：
影响因子：
3.8
通讯作者：
Povirk LF
中科院分区：
文献类型：
--
作者：
Chalasani SL;Kawale AS;Akopiants K;Yu Y;Fanta M;Weinfeld M;Povirk LF
多核苷酸激酶/磷酸酶(PNKP)与DNA双链断裂的非同源末端连接(NHEJ)有关。为了评估PNKP缺乏对3‘-磷酸末端DSB的NHEJ的影响，使用CRISPR/Cas9产生了HCT116和HeLa细胞的PNKP缺乏的衍生物。在这两种细胞系中，PNKP缺乏不仅对电离辐射敏感，而且对新卡宾抑素(NCS)敏感，NCS特异性地诱导DSB带有突起的3‘-磷酸末端。此外，与野生型(WT)相比，NCS诱导的DSB(被检测为53BP1灶)在PNKP−/−HCT116细胞中更持久。令人惊讶的是，缺乏PNKP的全细胞和细胞核提取物在生化上都能从合成的DSB底物中去除突起和凹陷的3‘-磷酸，尽管效率比WT提取物低得多，这表明有一种替代的3’-磷酸酶。连接介导的聚合酶链式反应结果显示，在NCS处理后5-15min，PNKP基因缺陷的HeLa细胞中3‘-磷酸末端和3’-羟基末端的DSB含量显著高于亲本细胞，但这种差异在1小时后消失。这些结果表明，尽管存在替代的3‘-磷酸酶，但由于3’-去磷酸化缺陷，PNKP的缺失显著地使细胞对3‘-磷酸终止的DSB敏感。
Polynucleotide kinase/phosphatase (PNKP) has been implicated in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). To assess the consequences of PNKP deficiency for NHEJ of 3′-phosphate-ended DSBs, PNKP-deficient derivatives of HCT116 and of HeLa cells were generated using CRISPR/CAS9. For both cell lines, PNKP deficiency conferred sensitivity to ionizing radiation as well as to neocarzinostatin (NCS), which specifically induces DSBs bearing protruding 3′-phosphate termini. Moreover, NCS-induced DSBs, detected as 53BP1 foci, were more persistent in PNKP−/− HCT116 cells compared to their wild-type (WT) counterparts. Surprisingly, PNKP-deficient whole-cell and nuclear extracts were biochemically competent in removing both protruding and recessed 3′-phosphates from synthetic DSB substrates, albeit much less efficiently than WT extracts, suggesting an alternative 3′-phosphatase. Measurements by ligation-mediated PCR showed that PNKP-deficient HeLa cells contained significantly more 3′-phosphate-terminated and fewer 3′-hydroxyl-terminated DSBs than parental cells 5-15 min after NCS treatment, but this difference disappeared by 1 hour. These results suggest that, despite presence of an alternative 3′-phosphatase, loss of PNKP significantly sensitizes cells to 3′-phosphate-terminated DSBs, due to a 3′-dephosphorylation defect.
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影响因子：
3.4
作者：
Alotaibi M;Sharma K;Saleh T;Povirk LF;Hendrickson EA;Gewirtz DA
通讯作者：
Gewirtz DA
影响因子：
3.5
作者：
Harris, Janelle L.;Jakob, Burkhard;Lavin, Martin F.
通讯作者：
Lavin, Martin F.
影响因子：
3.5
作者：
Black SJ;Kashkina E;Kent T;Pomerantz RT
通讯作者：
Pomerantz RT
DOI：
10.1016/0006-291x(74)90449-5
发表时间：
1974-01-01
影响因子：
3.1
作者：
BEERMAN, TA;GOLDBERG, IH
通讯作者：
GOLDBERG, IH
影响因子：
11.2
作者：
Karimi-Busheri, Feridoun;Rasouli-Nia, Aghdass;Weinfeld, Michael
通讯作者：
Weinfeld, Michael