An actinomycin-sensitive endonuclease associated with DNA synthesis in Tradescantia nuclei

A DNA synthetic system containing isolated Tradescantia pollen grain nuclei as a source of enzymes and endogenous DNA was used to study the effects of actinomycin-D (AM) with poly d(A-T) as exogenous primer. Poly d(A-T) causes a 10- to 50-fold increase in TMP incorporation which is depressed by 45 to 70% when 40 μM AM is present. The inhibition cannot be ascribed to interference with template function since poly d(A-T) does not bind AM. Inhibition of the synthesis of enzymes required for DNA replication is also not a possibility. In addition, AM has no direct effect on DNA polymerase. Exonuclease activity is inhibited from 0–30%, indicating that poly d(A-T) does not become the preferred substrate when the endogenous DNA is protected by AM. With paper and column chromatography, an endonuclease activity was demonstrated that is apparently responsible for the production of a range of oligonucleotides from poly d(A-T) of initial molecular weight ⩾5 × 10 6. These oligomers have a maximum molecular weight of ~2 × 10 4, and can serve as primers for DNA polymerase with the same efficiency as the higher molecular weight poly d(A-T). The endonuclease activity is inhibited 40 to 60% by AM. Many parallels are observed between the kinetics of DNA polymerase and those of the endonuclease in the presence and absence of AM. Both activities show the same degree of AM inhibition and both respond similarly to a change in pH. Furthermore, as saturating concentrations of poly d(A-T) are approached, the AM inhibition of TMP incorporation approaches zero. This AM effect is therefore considered to be a result of the reduction in number of available 3′-OH primer ends for DNA polymerase.