Translesion DNA synthesis in the dihydrofolate reductase domain of UV-irradiated CHO cells.
Translesion DNA synthesis in the dihydrofolate reductase domain of UV-irradiated CHO cells.
复制标题
紫外线照射的 CHO 细胞二氢叶酸还原酶结构域中的跨损伤 DNA 合成。
DOI：
10.1021/bi00144a021
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Hanawalt,PC
中科院分区：
文献类型：
--
作者：
Spivak,G;Hanawalt,PC
1992年4月27日摘要：这些研究记录了链特异性DNA修复与活性基因转录的耦合，将复制的DNA排除在分析之外。然而，环丁烷嘧啶二聚体（CPD）诱导的紫外线（UV）仍然存在于大部分的中国仓鼠卵巢（CHO）细胞的基因组中。在模板受损的情况下允许DNA复制发生的机制知之甚少。我们研究了CPD在复制DNA中二氢叶酸还原酶基因（DHFR）结构域中的分布。在UV照射后，在BrdUrd存在下孵育CHO Bll细胞;通过在CsCl中等密度沉降将复制的DNA与未复制的DNA分离，然后在碱性CsCl中解析亲本链和子链。我们确定了DHFR基因的14-kb Kpn I片段的部分，该片段对CPD特异性酶T4内切核酸酶V的消化具有抗性。在亲本和未复制的DNA中，约80%的CPD在24小时内从转录链上被去除，而约20%的CPD在24小时内从DHFR的非转录链上被去除。在一个15-kb的Kpnl片段，含有复制起点，并位于下游的DHFR-15 kb，我们发现非常低的修复水平，无论它是否已被复制。我们在分析的任一片段的子链中均未检测到CPD。这些结果表明，在没有显著水平的修复或链交换的情况下，复制叉可以穿过受损的DNA，并且CPD的修复不受这些细胞中复制的影响。用紫外线（UV）照射细胞 * 1会在其DNA中产生损伤，这些损伤已被证明能短暂抑制复制。中等剂量的紫外线（254 nm）产生的主要病变是环丁烷嘧啶二聚体（CPD）;其他较不常见的病变包括6-4嘧啶-嘧啶酮光产物[（6，4）PP]和氧化产物，如胸腺嘧啶二醇。
Revised Manuscript Received April 27, 1992 abstract: The studies that document the coupling of strand-specific DNA repair to transcription of active genes exclude replicated DNA from theanalysis. Yet cyclobutane pyrimidine dimers (CPD) induced by ultraviolet light (UV) persist in most of the genome in surviving Chinese hamster ovary (CHO) cells. The mechanisms that allow DNA replication to occur in the presence of damaged templates are poorly understood. We have investigated the distribution of CPD in the dihydrofolate reductase gene (DHFR) domain in replicated DNA. CHO Bll cells were incubated in the presence of BrdUrd after UV irradiation; the replicated DNA was separated from the unreplicated DNA by isopycnic sedimentation in CsCl, and then the parental and daughter strands were resolved in alkaline CsCl. We determined the fraction of a 14-kb Kpn\fragment of the DHFR gene that was resistant to digestion by T4 endonuclease V, a CPD-specific enzyme. In both parental and unreplicated DNA,~ 80% of the CPD were removed from the transcribed strands while~ 20% were removed from the nontranscribed strands of DHFR within 24 h. In a 15-kb Kpnl fragment that contains an origin of replication and is located—15 kb downstream of DHFR, we found very low repair levels, whether it had been replicated or not. We detected no CPD in the daughter strands of either fragment analyzed. These results suggest that the replication forks can move through the damaged DNA in the absence of significant levels of repair or strand exchange and that the repair of CPD is not affected by replication in these cells.The irradiation of cells with ultraviolet light (Uv)* 1 produces lesions in their DNA which have been shown totransiently inhibit replication. The primary lesions produced by moderate doses of UV (254 nm) are cyclobutane pyrimidine dimers (CPD); other, less frequent lesions include 6-4 pyrimidine-pyrimidone photoproducts [(6, 4) PP] and oxidation products such as thymine glycols.