Wang Y C, Hsu M T
Institute of Toxicology, Chung Shan Medical and Dental College, Taichung, Taiwan, Republic of China.
Nucleic Acids Res. 1996 Aug 15;24(16):3149-57. doi: 10.1093/nar/24.16.3149.
To study the mechanism by which ultraviolet (UV) light inhibits DNA replication, we examined the effects of UV 254 nm irradiation on the replication of simian virus 40 (SV40) DNA and SV40-based plasmid in monkey cells. The study was designed to determine the relative contributions made by inhibition of replication initiation and chain elongation to the immediate inhibition of DNA replication following UV irradiation. We used two-dimensional neutral-alkaline electrophoresis to examine the behaviour of replication intermediates unambiguously. Kinetic analysis using this technique showed that initiation of replication started to decline at 15 min post-irradiation. When the pulse label incorporated in SV40 replication intermediates before irradiation was chased for 1 h, most of the label was found in mature Form I and II molecules. This indicated that replication elongation took place on damaged template. We also used a transfection technique to show that heavily irradiated plasmids replicated efficiently in unirradiated transfected cells. By the transfection technique, we observed that UV irradiation of host cells dose-dependently inhibited replication of transfected non-irradiated plasmids, suggesting that the inhibition of DNA replication is due to a global change in cellular physiology induced by UV. This change was also apparent from poor staining of the chromatin by fluorescent-DNA-binding dyes immediately after UV irradiation of intact cells. We conclude that a significant fraction of chain elongation proceeds on damaged templates and DNA replication during the acute response of cells irradiated with UV is mainly controlled by the inhibition of replication initiation.
为了研究紫外线（UV）抑制DNA复制的机制，我们检测了254nm紫外线照射对猴细胞中猿猴病毒40（SV40）DNA及基于SV40的质粒复制的影响。该研究旨在确定复制起始抑制和链延伸抑制对紫外线照射后DNA复制即时抑制的相对贡献。我们使用二维中性-碱性电泳明确检测复制中间体的行为。使用该技术的动力学分析表明，照射后15分钟复制起始开始下降。当对照射前掺入SV40复制中间体的脉冲标记进行1小时追踪时，大部分标记物存在于成熟的I型和II型分子中。这表明在受损模板上发生了复制延伸。我们还使用转染技术表明，重度照射的质粒在未照射的转染细胞中能有效复制。通过转染技术，我们观察到宿主细胞的紫外线照射剂量依赖性地抑制转染的未照射质粒的复制，这表明DNA复制的抑制是由于紫外线诱导的细胞生理全局变化。完整细胞紫外线照射后立即用荧光DNA结合染料对染色质染色不佳也表明了这种变化。我们得出结论，相当一部分链延伸在受损模板上进行，紫外线照射细胞急性反应期间的DNA复制主要受复制起始抑制的控制。
