Depletion of poly(ADP-ribose) polymerase by antisense RNA expression results in a delay in DNA strand break rejoining.
Depletion of poly(ADP-ribose) polymerase by antisense RNA expression results in a delay in DNA strand break rejoining.
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DOI：
10.1016/s0021-9258(18)42347-2
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发表时间：
1992-06
期刊：
影响因子：
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通讯作者：
R. Ding;Yves PommierS;Veronica H. Kang;Mark Smulsonj
中科院分区：
文献类型：
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作者：
R. Ding;Yves PommierS;Veronica H. Kang;Mark Smulsonj
为了进一步了解核蛋白聚ADP核糖基化修饰的生物学作用，测定了多聚ADP核糖聚合酶（PADPRP）反义RNA在HeLa细胞中的诱导表达效果。将小鼠乳腺肿瘤病毒（MMTV）启动子插入到PADPRP cDNA的反义序列上游，构建了重组表达质粒。反义RNA的表达受到严格控制，在不存在诱导剂的情况下，对细胞生长的影响可忽略不计。与先前描述的PADPRP的稳定性（体内半衰期至少为2天）一致，在地塞米松诱导反义RNA表达后需要48-72小时，以使通常存在于HeLa细胞中的丰富浓度的PADPRP减少大于80%。诱导反义RNA表达介导的内源性PADPRP耗竭通过以下方式确定：（i）通过北方分析评估的细胞中反义转录物的逐步合成;（ii）酶活性降低80%;和（iii）完整细胞中的免疫印迹和免疫组织化学分析证实的PADPRP蛋白的细胞含量降低90%以上。在耗尽PADPRP的细胞中监测几个生物学参数。脱氧核糖核酸酶I敏感性评估显示，PADPRP耗竭细胞的染色质结构发生改变。细胞形态也发生了变化，多核聚集体在诱导反义RNA表达后72小时明显。耗尽PADPRP的细胞不能开始受损DNA的DNA链断裂连接。然而，DNA修复能力在稍后的时间段重新建立，表明PADPRP可能有助于改变染色质结构，最初发生在DNA链断裂重新连接和细胞核中的酶的浓度超过DNA修复/复制的要求。
The effects of inducible expression of poly(ADP-ribose) polymerase (PADPRP) antisense RNA in HeLa cells were determined in order to gain further insight into the biological roles of the poly(ADP-ribosyl)ation modification of nuclear proteins. A recombinant expression plasmid was prepared with the mouse mammary tumor virus (MMTV) promoter upstream of the antisense-oriented PADPRP cDNA. Expression of the antisense RNA was under strict control, with negligible effects on cell growth being apparent in the absence of inducer. Consistent with the previously described stability of PADPRP (half-life of at least 2 days, in vivo), 48-72 h were required after induction of antisense RNA expression by dexamethasone for the abundant concentration of PADPRP, normally present in HeLa cells, to be reduced by greater than 80%. The depletion of endogenous PADPRP as mediated by induced antisense RNA expression was established by: (i) a progressive synthesis of antisense transcripts in cells as assessed by Northern analysis; (ii) an 80% decrease in activity of the enzyme; and (iii) a greater than 90% reduction in the cellular content of PADPRP protein, as demonstrated by both immunoblotting and immunohistochemical analysis in intact cells. Several biological parameters were monitored in cells depleted of PADPRP. The chromatin of PADPRP-depleted cells was shown to have an altered structure as assessed by deoxyribonuclease I susceptibility. Cell morphology was also altered, with multinucleated aggregates being evident 72 h after induction of antisense RNA expression. Cells depleted of PADPRP were not able to commence DNA strand break joining of damaged DNA. However, DNA repair capacity was re-established at later time periods, indicating that PADPRP may contribute to alterations in chromatin structure that occur initially in DNA strand break rejoining and that the concentration of the enzyme in nuclei exceeds the requirement for DNA repair/replication.