Mezzina M, Menck C F, Courtin P, Sarasin A
Laboratory of Molecular Mutagenesis, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
J Virol. 1988 Nov;62(11):4249-58. doi: 10.1128/JVI.62.11.4249-4258.1988.
The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. (i) In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. (ii) In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Cairns-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. (iii) Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules. These results are in agreement with and reinforce the model in which UV lesions are a barrier to the replication fork movement when present in the template for the leading strand; when lesions are in the template for the lagging strand they inhibit synthesis or completion of Okazaki fragments, leaving gaps opposite the lesion. Moreover, cellular DNA repair-linked endonucleolytic activity may induce double-stranded breaks in the blocked region of the replication forks, resulting in the tailed structures observed in viral DNA molecules obtained from excision repair-proficient cell lines.
在感染了猴病毒40的猴和人细胞中，研究了暴露于紫外线（254纳米）下对哺乳动物DNA复制进行体内抑制的分子机制。通过电子显微镜和蔗糖梯度对病毒DNA进行分析，证实紫外线诱导损伤的存在严重阻碍了DNA合成，因此紫外线照射抑制了复制中间体（RI）转化为完全复制的I型DNA。当通过电子显微镜观察时，这些受阻的RI分子呈现出几个特殊特征。（i）在具有切除修复能力的猴和人细胞中，它们由具有双链尾巴的双链环状DNA组成，尾巴的大小与嘧啶二聚体的平均间距相对应，这是由二聚体特异性的T4内切核酸酶V测定的。（ii）在患有色素性干皮病患者的切除修复缺陷的人细胞中，紫外线照射的RI呈现出类似凯恩斯结构，类似于从未经照射的感染细胞中获得的复制分子所观察到的结构。（iii）在紫外线照射的RI分子的复制部分可见单链缺口；当用特定的单链DNA结合蛋白（如噬菌体T4基因32产物）探测时，这些区域可以被检测到并与双链DNA明显区分开来。与紫外线照射的RI分子中存在缺口一致，与未受损分子相比，在中性蔗糖梯度中分析时，单链特异性S1核酸酶消化会导致它们的沉降特性发生变化。这些结果与以下模型一致并加强了该模型：当紫外线损伤存在于前导链的模板中时，它们是复制叉移动的障碍；当损伤存在于滞后链的模板中时，它们会抑制冈崎片段的合成或完成，在损伤相对处留下缺口。此外，与细胞DNA修复相关的内切核酸酶活性可能会在复制叉的受阻区域诱导双链断裂，从而导致在从具有切除修复能力的细胞系中获得的病毒DNA分子中观察到的带尾结构。
