Matsumoto S S, Yu J, Yu A L
Department of Pediatrics, University of California, San Diego, La Jolla 92093.
J Biol Chem. 1988 May 25;263(15):7153-8.
Deoxyadenosine plus deoxycoformycin (dCf) causes increased DNA breaks in lymphoid cells. This study explored the possible inhibition of repair synthesis of DNA by dAdo plus dCf as a cause of DNA breakage. It was shown that DNA breaks accumulated in a human T-lymphoblast cell line, CCRF-CEM, following incubation with dAdo plus dCf and were not fully repaired 20 h after their removal. Analysis of the density distribution of radiolabeled DNA on alkaline CsCl gradient showed that incubation of CCRF-CEM cells with dAdo plus dCf caused inhibition of semiconservative, but not repair synthesis of DNA. Semiconservative synthesis of DNA was also inhibited in CCRF-CEM nuclei isolated from cells pretreated with dAdo and dCf, suggesting damage to DNA replicative machinery. However, no such inhibition was observed in the nuclei of a similarly treated CCRF-CEM mutant that was deficient in adenosine kinase and deoxycytidine kinase. This suggests that dAdo must be phosphorylated in intact cells to exert its effect. Using [3H]dTTP incorporation in isolated CCRF-CEM nuclei to measure DNA synthesis, it was found that a high concentration (greater than 100 microM) of dATP inhibits semiconservative but not repair synthesis of DNA. The present studies thus indicate that accumulation of DNA strand breaks induced by dAdo plus dCf is not the consequence of inhibition of repair DNA synthesis. This implies the mechanism may involve perturbation of DNA ligation or activation of a certain process which causes DNA strand breaks. In addition, dATP may interfere with some steps of semiconservative DNA synthesis, but not the repair synthesis of DNA.
脱氧腺苷加脱氧助间型霉素（dCf）会导致淋巴细胞中的DNA断裂增加。本研究探讨了脱氧腺苷加脱氧助间型霉素对DNA修复合成的可能抑制作用，以此作为DNA断裂的一个原因。结果显示，在人T淋巴母细胞系CCRF - CEM中，用脱氧腺苷加脱氧助间型霉素孵育后，DNA断裂会累积，且在去除药物后20小时仍未完全修复。对放射性标记的DNA在碱性CsCl梯度上的密度分布分析表明，用脱氧腺苷加脱氧助间型霉素孵育CCRF - CEM细胞会抑制DNA的半保留合成，但不抑制DNA的修复合成。从用脱氧腺苷和脱氧助间型霉素预处理过的细胞中分离出的CCRF - CEM细胞核内，DNA的半保留合成也受到抑制，这表明DNA复制机制受到了损害。然而，在腺苷激酶和脱氧胞苷激酶缺陷的类似处理的CCRF - CEM突变体的细胞核中未观察到这种抑制作用。这表明脱氧腺苷必须在完整细胞中磷酸化才能发挥其作用。利用[3H]胸苷三磷酸掺入分离出的CCRF - CEM细胞核中来测量DNA合成，发现高浓度（大于100 microM）的脱氧三磷酸腺苷抑制DNA的半保留合成，但不抑制DNA的修复合成。因此，目前的研究表明，脱氧腺苷加脱氧助间型霉素诱导的DNA链断裂的积累不是抑制修复性DNA合成的结果。这意味着其机制可能涉及DNA连接的扰动或导致DNA链断裂的某个过程的激活。此外，脱氧三磷酸腺苷可能会干扰DNA半保留合成的某些步骤，但不会干扰DNA的修复合成。
