DNA核苷酸切除修复合成依赖于脱氧核苷三磷酸池大小的扰动

Edward J. Katz, Michael A. Sirover
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引用次数: 11

摘要

我们研究了脱氧核苷三磷酸(dNTP)池大小的波动对DNA修复的影响,反过来,DNA修复对dNTP池大小的影响。在融合的正常人皮肤成纤维细胞中,通过[3H]胸苷形成[3H]TTP来定量dNTP池的大小;在紫外光照射下,用修复复制法检测DNA修复。HPLC分析表明,[3H]TTP池在加入[3H]胸苷后30分钟内形成,并在接下来的6小时内保持相对稳定。加入2-10 mM的羟基脲(HU)导致[3H]TTP池逐渐增加2 - 4倍,因为HU抑制了DNA合成,但不抑制TTP的产生。暴露于20 M/m2的细胞和未辐射的对照组的[3H]TTP池大小没有差异,尽管前者的dna修复合成很容易定量。尽管与最初的研究相比,修复复制方案导致[3H]TTP池的大小减少了8 - 10倍,但仍观察到这一结果。在紫外线切除修复研究中,羟基脲的存在并没有改变由于修复体复制而并入亲本DNA的[3H]胸苷5 '-单磷酸酯的特异性活性。这些结果表明,脱氧核苷三磷酸池的波动不会限制人类细胞中切除-修复合成的程度,并表明DNA核苷酸切除-修复合成不会显著减少[3H]TTP池的大小。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
DNA nucleotide excision-repair synthesis is dependent of pertubations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 M/m2 and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool.

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