Sinha N K, Snustad D P
J Bacteriol. 1972 Dec;112(3):1321-4. doi: 10.1128/jb.112.3.1321-1334.1972.
The effects of hydroxyurea on Escherichia coli B/5 physiology (increases in cell mass, number of viable cells, and deoxyribonucleic acid [DNA], RNA, and protein concentrations) were studied in an attempt to find a concentration that completely inhibits DNA synthesis and increase in number of viable cells but has little or no effect on other metabolic processes. These conditions were the most closely approached at an hydroxyurea concentration of 0.026 to 0.033 m. A concentration of 0.026 or 0.033 m was used in subsequent experiments to study the site(s) of inhibition of DNA synthesis in E. coli B/5 by hydroxyurea. Hydroxyurea at a concentration of 10(-2)m was found to inhibit ribonucleoside diphosphate reductase activity completely in crude extracts of E. coli. The synthesis of deoxyribonucleotides was greatly reduced when E. coli cells were grown in the presence of 0.033 m hydroxyurea. Studies on the acid-soluble DNA precursor pools showed that hydroxyurea causes a decrease in the concentration of deoxyribonucleoside diphosphates and deoxyribonucleoside triphosphates and an increase in the total concentration of ribonucleotides. Sucrose density gradient sedimentation of DNA from cells treated with 0.026 m hydroxyurea for 30 min indicated that at this concentration hydroxyurea induces no detectable single- or double-strand breaks. In addition, both replicative and repair syntheses of DNA were found to occur normally in toluene-treated cells in the presence of relatively high concentrations of hydroxyurea. Pulse-chase studies showed that deoxyribonucleotides synthesized prior to the addition of hydroxyurea to cells are utilized normally for DNA synthesis in the presence of hydroxyurea. On the basis of these observations, we have concluded that the primary, if not the only, site of inhibition of DNA synthesis in E. coli B/5 by low concentrations of hydroxyurea is the inhibition of the enzyme ribonucleoside diphosphate reductase.
研究了羟基脲对大肠杆菌B/5生理学的影响（细胞质量增加、活细胞数量增加以及脱氧核糖核酸[DNA]、RNA和蛋白质浓度增加），试图找到一种浓度，该浓度能完全抑制DNA合成并增加活细胞数量，但对其他代谢过程几乎没有影响。在羟基脲浓度为0.026至0.033 m时最接近这些条件。在随后的实验中，使用0.026或0.033 m的浓度来研究羟基脲对大肠杆菌B/5中DNA合成的抑制位点。发现浓度为10(-2)m的羟基脲能完全抑制大肠杆菌粗提物中的核糖核苷二磷酸还原酶活性。当大肠杆菌细胞在0.033 m羟基脲存在下生长时，脱氧核糖核苷酸的合成大大减少。对酸溶性DNA前体库的研究表明，羟基脲会导致脱氧核糖核苷二磷酸和脱氧核糖核苷三磷酸浓度降低，核糖核苷酸总浓度增加。用0.026 m羟基脲处理30分钟的细胞的DNA蔗糖密度梯度沉降表明，在此浓度下羟基脲不会诱导可检测到的单链或双链断裂。此外，发现在相对高浓度的羟基脲存在下，经甲苯处理的细胞中DNA的复制和修复合成均正常发生。脉冲追踪研究表明，在向细胞中添加羟基脲之前合成的脱氧核糖核苷酸在羟基脲存在下正常用于DNA合成。基于这些观察结果，我们得出结论，低浓度羟基脲对大肠杆菌B/5中DNA合成的主要抑制位点（如果不是唯一的位点）是对核糖核苷二磷酸还原酶的抑制。
