A SPECIALIZED FORM OF CHROMOSOMAL DNA-DEGRADATION INDUCED BY THYMIDYLATE STRESS IN MOUSE FM3A CELLS
A SPECIALIZED FORM OF CHROMOSOMAL DNA-DEGRADATION INDUCED BY THYMIDYLATE STRESS IN MOUSE FM3A CELLS
复制标题
DOI：
10.1016/0027-5107(88)90086-3
复制
发表时间：
1988-07-01
期刊：
影响因子：
--
通讯作者：
SENO, T
中科院分区：
文献类型：
--
作者：
AYUSAWA, D;ARAI, H;SENO, T
经5-氟-2‘’-脱氧尿嘧啶核苷处理的胸苷合成酶阴性突变小鼠细胞或其亲本FM3A细胞经5-氟-2‘-脱氧尿苷处理后，产生50-200kb的DNA片段，经脉冲场琼脂糖凝胶电泳法测定，在100kb处有一峰。胸苷应激后DNA片段的积累是时间依赖的，但它们的大小分布在这两种情况下都没有改变。DNA合成的脉冲标记显示，染色体DNA断裂的区域似乎仅限于DNA复制正在进行的区域。埃美汀是一种蛋白质合成的抑制剂，在胸苷应激期间，当存在时，它会阻止DNA片段的积累。用上述两种方法中的任何一种方法从胸腺酸应激细胞中制备的无细胞提取物都能够降解从体外正常生长的细胞中制备的染色质中的DNA。产生的DNA片段相似，但与体内产生的DNA片段相比，其大小分布略有扩大。在体外反应中产生的片段的更广泛的分布变得更接近于在体内获得的模式，当ATP和4个脱氧核苷酸被加入到反应中时。
Thymidylate synthase-negative mutant mouse cells starved of thymidine or their parental FM3A cells treated with 5-fluoro-2''-deoxyuridine produced DNA fragments ranging from 50 to 200 kilobase pairs with a peak at 100 kb in length as determined by pulsed-field agarose gel electrophoresis. Accumulation of the DNA fragments following such thymidylate stress was time-dependent but their size distribution did not change in either case. Regions of the chromosomal DNA breaks seemed to be restricted to those where DNA replication was in progress as shown by pulse-labeling of the DNA synthesis. Emetine, an inhibitor of protein synthesis, blocked the accumulation of the DNA fragments when present during thymidylate stress. Cell-free extracts prepared from the thymidylate-stressed cells derived by either of the above means were capable of degrading DNA in chromatins prepared from normally growing cells in vitro. The resulting DNA fragments were similar but with a somewhat broader size distribution compared to those produced in vivo. The broader distribution of the fragments produced in the in vitro reaction became closer to the pattern obtained in vivo when ATP and 4 deoxyribonucleotides were added to the reaction.