Topoisomerase II cleavable complex formation within DNA loop domains.
Topoisomerase II cleavable complex formation within DNA loop domains.
复制标题
DNA 环结构域内拓扑异构酶 II 可切割复合物的形成。
DOI：
10.1016/s0006-2952(00)00309-9
复制
发表时间：
2000
影响因子：
5.8
通讯作者：
Fernandes,DJ
中科院分区：
文献类型：
--
作者：
Lambert,JM;Fernandes,DJ
测定VM-26（替尼泊苷）稳定的可裂解复合物在与核基质结合的DNA环内的分布，以进一步了解VM-26抑制DNA合成的模式。与CEM细胞的高盐溶性非基质蛋白相比，[3 H]VM-26的共价结合是每毫克核基质蛋白的9倍。该比率从CEM细胞的9倍下降到耐药VM-1/C2细胞的4倍，其具有减少的核基质DNA拓扑异构酶IIα。VM-26诱导与活跃复制的基质DNA形成可切割复合物的频率的浓度依赖性增加。VM-26浓度为25 μM时，复制型基质DNA中每106 bp出现32 ± 2个复合物（SEM），而基质部分中每106 bp非复制型DNA出现13 ± 2个复合物（SEM）。VM-26在高达25 μM的浓度下，在各种非基质DNA结构域中每106 bp稳定不到3个复合物，因为非基质DNA包含远离基质结合复制位点的DNA环结构域。在耐药VM-1/C2细胞的基质和非基质DNA结构域中检测到可切割复合物形成的频率可以忽略不计。与未处理的对照细胞相比，VM-26诱导新生DNA在CEM细胞的核基质部分中的积累，但减少了新生DNA在非基质部分中的量。基质复制DNA上广泛的可裂解复合物形成使大多数复制叉停滞在核基质上的复制位点的Ikb内。结果提供证据表明，新生的DNA结合到核基质是VM-26可切割的复合物形成的一个重要位点，这些复合物通过阻断新生的DNA远离核基质上的复制位点的运动来抑制DNA合成。
The distribution of VM-26 (Teniposide)-stabilized cleavable complexes within DNA loops bound to the nuclear matrix was determined to provide further insights into the mode of DNA synthesis inhibition by VM-26. Covalent binding of [3H]VM-26 was 9-fold greater per milligram of nuclear matrix protein compared with high salt-soluble nonmatrix protein of CEM cells. The ratio declined from 9-fold in CEM cells to 4-fold in drug-resistant VM-1/C2 cells, which have decreased nuclear matrix DNA topoisomerase IIα. VM-26 induced a concentration-dependent increase in the frequency of cleavable complex formation with actively replicating matrix DNA. At 25 μM VM-26, the frequency was 32 ± 2 (SEM) complexes per 106bp of replicating matrix DNA compared with 13 ± 2 (SEM) complexes per 106bp of nonreplicating DNA in the matrix fraction. VM-26 at concentrations as high as 25 μM stabilized less than 3 complexes per 106bp in the various nonmatrix DNA domains, since the nonmatrix DNA comprises the DNA loop domains that are distal to the matrix-bound replication sites. A negligible frequency of cleavable complex formation was detected in both the matrix and nonmatrix DNA domains of drug-resistant VM-1/C2 cells. Compared with untreated control cells, VM-26 induced an accumulation of nascent DNA in the nuclear matrix fraction of CEM cells but decreased the amount of nascent DNA in the nonmatrix fraction. The extensive cleavable complex formation on matrix replicating DNA stalled most of the replication forks within 1 kb of the replication sites on the nuclear matrix. The results provide evidence that nascent DNA bound to the nuclear matrix is an important site of VM-26 cleavable complex formation, and that these complexes inhibit DNA synthesis by blocking the movement of nascent DNA away from replication sites on the nuclear matrix.