Topoisomerase II cleavable complex formation within DNA loop domains

Abstract

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 10 6 bp of replicating matrix DNA compared with 13 ± 2 (SEM) complexes per 10 6 bp of nonreplicating DNA in the matrix fraction. VM-26 at concentrations as high as 25 μM stabilized 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 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.