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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.
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A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

机译：人类聚（ADP-核糖）聚合酶的显性负突变体影响细胞恢复，细胞​​凋亡和DNA损伤后的染色单体交换。

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摘要

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.
机译：聚（ADP-核糖）聚合酶[PARP; NAD + ADP-核糖基转移酶； NAD +：聚（腺苷二磷酸-D-核糖基）受体ADP-D-核糖基转移酶，EC 2.4.2.30]是锌依赖性的真核DNA结合蛋白，可特异性识别各种遗传毒性剂产生的DNA链断裂。为了研究这种酶的生物学功能，我们建立了稳定的HeLa细胞系，该细胞组成性地产生人PARP（PARP-DBD）的46 kDa DNA结合域，从而导致对驻地PARP活性的反式抑制。作为对照，构建了细胞系，产生了DBD的点突变形式，该形式在体外对DNA没有亲和力。 PARP-DBD的表达仅对未受损的细胞产生轻微影响，但对用遗传毒性剂处理的细胞却产生了严重后果。用低剂量的N-甲基-N'-硝基-N-亚硝基胍（MNNG）暴露表达野生型（wt）或突变的PARP-DBD的细胞系会导致其倍增时间增加，即G2 + M积累，并显着降低细胞存活率。但是，UVC辐照对表达PARP-DBD的细胞系的细胞生长或活力没有优先的影响。这些用MNNG处理的PARP-DBD表达细胞呈现出特征性的核小体DNA阶梯，这是细胞凋亡导致细胞死亡的标志之一。此外，这些细胞表现出染色体不稳定性，如自发性和MNNG诱导的姐妹染色单体交换的较高频率所证明。出人意料的是，产生突变DBD的品系与产生wt DBD的品系具有相同的行为，这表明显性负突变体的作用机理所涉及的不仅仅是其DNA结合功能。总而言之，这些结果强烈表明，PARP是哺乳动物细胞中G2检查点的一个元素。

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