Synchronous Block in DNA Synthesis Initiation with Change in Chromatin Topology Mediated by VP 16

Abstract

The impact of chromatin topology on the DNA synthetic process was studied in the human squamous-cell carcinoma cell line SQ-20B. A 1-h exposure ≤10 μM VP16 produced an increase in DNA supercoil tension, measured by recording laser light scatter from salt-extracted nuclei. This change was precisely paralleled by a decrease in DNA synthesis. The effects on both DNA supercoiling and DNA synthesis were suppressed at VP16 concentrations between 10 and 20 μM. The changes in DNA supercoiling and synthesis at VP16 concentrations ≤10 μM were eliminated by coincubation with mimosine, a DNA synthesis initiator poison. We conclude that brief exposure to low concentrations of VP16 disturbs the balance of torsional energy within discrete replicon domains by affecting normal topoisomerase II activity at sites of replication initiation. The resultant increase in negative supercoil tension mediates a topologic checkpoint, limiting the initiation of DNA synthesis. Such a checkpoint may be a common pathway for control, both during the normal replicative cycle and subsequent to DNA damage.