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首页> 外文期刊>Molecular and Cellular Biology >Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts.
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Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts.

机译：在人和猴细胞提取物中复制和诱变紫外线破坏的DNA模板。

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We have used in vitro DNA replication systems from human HeLa cells and monkey CV-1 cells to replicate a UV-damaged simian virus 40-based shuttle vector plasmid, pZ189. We found that replication of the plasmid was inhibited in a UV fluence-dependent manner, but even at UV fluences which caused damage to essentially all of the plasmid molecules some molecules became completely replicated. This replication was accompanied by an increase (up to 15-fold) in the frequency of mutations detected in the supF gene of the plasmid. These mutations were predominantly G:C-->A:T transitions similar to those observed in vivo. Treatment of the UV-irradiated plasmid DNA with Escherichia coli photolyase to reverse pyrimidine cyclobutane dimers (the predominant UV-induced photoproduct) before replication prevented the UV-induced inhibition of replication and reduced the frequency of mutations in supF to background levels. Therefore, the presence of pyrimidine cyclobutane dimers in the plasmid template appears to be responsible for both inhibition of replication and mutation induction. Further analysis of the replication of the UV-damaged plasmid revealed that closed circular replication products were sensitive to T4 endonuclease V (a pyrimidine cyclobutane dimer-specific endonuclease) and that this sensitivity was abolished by treatment of the replicated DNA with E. coli photolyase after replication but before T4 endonuclease treatment. These results demonstrate that these closed circular replication products contain pyrimidine cyclobutane dimers. Density labeling experiments revealed that the majority of plasmid DNA synthesized in vitro in the presence of bromodeoxyuridine triphosphate was hybrid density whether or not the plasmid was treated with UV radiation before replication; therefore, replication of UV-damaged templates appears to occur by the normal semiconservative mechanism. All of these data suggest that replication of UV-damaged templates occurs in vitro as it does in vivo and that this replication results in mutation fixation.
机译：我们已经使用了来自人类HeLa细胞和猴CV-1细胞的体外DNA复制系统来复制基于紫外线损伤的猿猴病毒40的穿梭载体质粒pZ189。我们发现质粒的复制以依赖于UV能量通量的方式被抑制，但是即使在影响了基本上所有质粒分子的UV能量通量下，一些分子也被完全复制。这种复制伴随着在质粒的supF基因中检测到的突变频率的增加（最多15倍）。这些突变主要是G：C-> A：T转换，与体内观察到的相似。复制前用大肠杆菌光解酶处理UV辐射的质粒DNA以逆转嘧啶环丁烷二聚体（主要的UV诱导的光产物）可防止UV诱导的复制抑制，并将supF突变的频率降低至背景水平。因此，质粒模板中嘧啶环丁烷二聚体的存在似乎是复制抑制和突变诱导的原因。进一步分析了受紫外线破坏的质粒的复制，发现闭合的环状复制产物对T4内切核酸酶V（嘧啶环丁烷二聚体特异性内切核酸酶）敏感，并且在用大肠杆菌光解酶处理复制的DNA后消除了这种敏感性。复制但在T4核酸内切酶治疗之前。这些结果表明，这些闭合的环状复制产物含有嘧啶环丁烷二聚体。密度标记实验表明，在三磷酸溴脱氧尿苷存在下，体外合成的大多数质粒DNA均为杂合密度，无论该质粒在复制前是否经过UV辐射处理。因此，紫外线破坏的模板的复制似乎是通过正常的半保守机制发生的。所有这些数据表明，紫外线破坏的模板的复制与体内的复制一样在体外发生，并且这种复制导致突变固定。

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