Topoisomerase II cleavable complex formation within DNA loop domains

The distribution of VM-26 (Teniposide)-stabilized cleavable complexes withi n DNA loops bound to the nuclear matrix was determined to provide further i nsights into the mode of DNA synthesis inhibition by VM-26. Covalent bindin g of [H-3]VM-26 was 9-fold greater per milligram of nuclear matrix protein compared with high salt-soluble nonmatrix protein of CEM cells. The ratio d eclined from 9-fold in CEM cells to 4-fold in drug resistant VM-1/C2 cells, which have decreased nuclear matrix DNA topoisomerase II alpha. VM-26 indu ced a concentration-dependent increase in the frequency of cleavable comple x formation with actively replicating matrix DNA. At 25 mu M VM-26, the fre quency was 32 +/- 2 (SEM) complexes per 10(6) bp of replicating matrix DNA compared with 13 +/- 2 (SEM) complexes per 10(6) bp of nonreplicating DNA i n the matrix fraction. VM-26 at concentrations as high as 25 mu M stabilize d less than 3 complexes per 10(6) bp in the various nonmatrix DNA domains, since the nonmatrix DNA comprises the DNA Loop domains that are distal to t he matrix-bound replication sites. A negligible frequency of cleavable comp lex 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 CE M 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 bond to the n uclear matrix is an important site of VM-26 cleavable complex formation, an d that these complexes inhibit DNA synthesis by blocking the movement of na scent DNA away from replication sites on the nuclear matrix. BIOCHEM PHARMA COL 60;1: 101-109, 2000. (C) 2000 Elsevier Science Inc.