The nuclear matrix model of DNA replication and organization will be utilized in the proposed studies to address (a) important effects of topoisomerase-active drugs on primary and higher order chromatin structure and (b) the cellular responses to these drug effects (e.g., DNA repair, chromatin disorganization) that influences drug action. The central hypothesis to be tested is that topoisomerase I and II poisons irreversibly damage DNA by preferentially inducing double-strand DNA breaks in replication forks on the nuclear matrix of human CEM cells. The cleaved replication forks then detach from the nuclear matrix, which interferes with the ability of the DNA protein kinase (DNA-PK) and replication protein A (RPA) systems to signal DNA damage control and replication fork repair. Preferential cleavage of its c-myc and beta-globin genes within various nuclear matrix DNA loop domains will be quantitated using the PCR-stop assay (Specific Aim 1). The studies described in Specific Aim 2a will extend these observations by evaluating some potentially important biological consequences of drug-induced detachment of c-myc and beta-globin Okazaki fragments and replicating DNA loops from the nuclear matrix.
Specific Aims 2 b and 2c are designed to assess some of the cellular responses to VM-26 and camptothecin induced damage to matrix-attached DNA loops. To evaluate the repair of VM-26 and camptothecin induced cleavage, the rates of reversal of drug-induced cleavage in replicating and nonreplicating c-myc and beta-globin genes will be determined in CEM and VM-26 resistant VM-1 cells. Additional experiments are designed to quantitate the degrees of drug-induced RPA32 hyperphosphorylation and Ku70-DNA-Pkcs binding to replicating and nonreplicating DNA. These studies will determine whether the extent of activation of the DNA-PK-RPA system for replication fork arrest and double strand DNA break repair is related to the degree of reversal of drug-induced DNA cleavage in these cell lines. The proposed studies represent a novel approach for providing insights into important biological effects of the topoisomerase I and topoisomerase II drugs as well as the cellular responses to these effects that may influence drug cytotoxicity.
| Otake, Yoko; Mims, Alice; Fernandes, Daniel J (2006) Merbarone induces activation of caspase-activated DNase and excision of chromosomal DNA loops from the nuclear matrix. Mol Pharmacol 69:1477-85 |
| Fernando, Lawrence P; Kurian, Philip J; Fidan, Melihat et al. (2002) Quantitation of gene-specific DNA damage by competitive PCR. Anal Biochem 306:212-21 |
| Lambert, J M; Fernandes, D J (2000) Topoisomerase II cleavable complex formation within DNA loop domains. Biochem Pharmacol 60:101-9 |
