The chromatin/DNA topology enzymes [i.e., poly(ADP-ribose) polymerase, and topoisomerases I and II] are considered to have important roles in DNA synthesis, RNA transcription, mitosis, and DNA-damage repair, and their functions are probably interactive. However, their specific role(s) in the repair of DNA damage from irradiation and drugs is uncertain and indeed controversial. The long term goals of this proposal are the elucidation of their interactive roles in the repair of DNA damage from ionizing radiation and mitomycin-C. The parental V79 hamster cells and two x-ray-sensitive mutants (irs-1 and irs-2) are being used and drug-resistant sublines have been developed to relatively specific inhibitors of these three enzymes [i.e., 3-aminobenzamide (3AB), camptothecin (Cp), and m-AMSA (mA) respectively]. The cellular radiation phenotype (cell survival) of the irs-2(3AB)R and irs-2(Cp)R cells has returned to the parental V79 phenotype while that of the V79(3AB)R is unchanged. The endogenous and x-ray-induced activity (incorp. of 3H-NAD) of the polymerase enzyme in the irs-2(3AB)R and irs-2(Cp)R have been elevated markedly above the parental-cell activity. Studies are being conducted on both the activity and enzyme content (Western blots) of the polymerase enzyme as well as that of the topo enzymes (the activity assays for the topos are alkaline and neutral filter elution respectively for Topo I and II after drug-induced DNA strand breaks). The question of gene-amplification (Southern blot analysis) and increased mRNA transcription or stabilization (Northern blot analysis and specific mRNA transcription rates) are being explored as a mechanism for the induced level of the polymerase enzyme. Also DNA damage induction and repair from mitomycin-C are being compared to the radiobiological data in these drug resistant lines to elevate the generality vs specificity of these altered phenotypes (i.e. both long-patch and short-patch repair). In summary, the critical metabolic aspects of these three nuclear enzymes are being studied """"""""in concert"""""""" as they relate to (possibly explain) the altered cellular radiation phenotype (resistance) in the (3AB)R and (Cp)R irs-2 sublines.
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