Molecular interactions between DNA, poly(ADP-ribose) polymerase, and histones.

S S Sastry,E Kun

doi:10.1016/s0021-9258(19)57332-x

Abstract

Molecular interactions between purified poly(ADP-ribose) polymerase, whole thymus histones, histone H1, rat fibroblast genomic DNA, and closed circular and linearized SV40 DNA were determined by the nitrocellulose filter binding technique. Binding of the polymerase protein or histones to DNA was augmented greatly when both the enzyme protein and histones were present simultaneously. The polymerase protein also associated with histones in the absence of DNA. The cooperative or promoted binding of histones and the enzyme to relaxed covalently closed circular SV40 DNA was greater than the binding to the linearized form. Binding of the polymerase to SV40 DNA fragments in the presence of increasing concentrations of NaCl indicated a preferential binding to two restriction fragments as compared to the others. Polymerase binding to covalently closed relaxed SV40 DNA resulted in the induction of superhelicity. The simultaneous influence of the polymerase and histones on DNA topology were more than additive. Topological constraints on DNA induced by poly(ADP-ribose) polymerase were abolished by auto ADP-ribosylation of the enzyme. Benzamide, by inhibiting poly(ADP-ribosylation), reestablished the effect of the polymerase protein on DNA topology. Polymerase binding to in vitro-assembled core particle-like nucleosomes was also demonstrated.

Highlights

From the Department of Pharmacology and the Cardiovascular Research Institute, The University of California, San Francisco, School of Medicine, San Francisco, California94143-0130
Polymerase protein or histonesDtNoA was augmented 2.4.2.30), a DNA-binding nuclear protein which greatly whenboththeenzymeproteinandhistones has been implicated in the control of a variety of cellular were present simultaneously
The poly(ADP-ribose) polymerase protein is not known to display, in terms of DNA binding, any DNA sequence specificity, enzymatic studies reveal that DNA molecules of differing sequence appear to stimulate enzymatic activity to various degrees [35].' rather thanexperimenting with specific DNA sequences as substrates for DNA binding, we first employed genomic DNAas binding ligand to provide broad-based DNA sequences

Summary

RESULTS

Promotion of Binding of Poly(ADP-ribose) Polymerase to DNA by Histones-Initially, the effects of whole thymus histones were determined because we could not predict if any of the histone subfractionsmight exhibit selectivity in terms of their interaction with DNA and the polymerase enzyme. If increasing amounts of 32P-labeledDNA is added to a constant amount of protein, aproportional increase in retention of 32Plabel on filters occurs (Fig. 1B). This indicates quantitative binding of both the polymerase and histones to restricted 14C DNA. The experimentalpoints in the upper z c curves of Fig. lA have been obtained by subtracting the percent radioactive material contributedby the protein present in constant amounftrsom the totalamount of radioactivity retained on the filter as a result of the binding of both proteins to DNA. Such an effect is notobserved with either the polymerase alone (Fig. L4, A) or when larger amounts of the polymerase are added with a small amount of histones (Fig. lA,0)

The Specificity of Histones in Promoting the Binding of

To rule out the possibility that the increased retention of

It is possible that thepreferential binding of the polymerase

DISCUSSION