Koichiro Yoshihara

Inhibition of DNA polymerase α, DNA polymerase β, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro☆

Incubation of DNA polymerase α1, DNA polymerase β, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furtheremore, the suppressed activities of DNA polymerase α, DNA polymerase β, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.

Enzyme-bound early product of purified poly(ADP-ribose) polymerase.

Abstract Bovine thymus poly(ADP-ribose) polymerase with a purity of 99% on a SDS-poly-acrylamide gel electrophoresis was able to initiate poly(ADP-ribose) synthesis without adding any exogenous acceptor protein to the reaction system. Analyses of the early reaction product synthesized without exogenous acceptor protein revealed that the product was oligo(ADP-ribose) with a mean chain length of 2.6 and was bound tightly to the enzyme protein. When the radioactive early reaction product was chased by incubating further with cold NAD + , ADP-ribose unit was found to be added to the terminal AMP-residue of the oligo(ADP-ribose) attached to the enzyme. The stability of the early reaction product in high concentration of salt, strong acid, sodium dodecyl sulfate, and urea strongly suggests a covalent nature of the binding of oligo(ADP-ribose) to the enzyme.

RNA Interference Directed against Poly(ADP-Ribose) Polymerase 1 Efficiently Suppresses Human Immunodeficiency Virus Type 1 Replication in Human Cells

ABSTRACT We established small interfering RNA (siRNA) directed against poly(ADP-ribose) polymerase 1 (PARP-1) that effectively reduces the expression of PARP-1 in two human cell lines. Established siRNA against PARP-1 significantly suppressed human immunodeficiency virus type 1 (HIV-1) replication, as well as the activation of the integrated HIV-1 long terminal repeat promoter. These results indicate that PARP-1 is required for efficient HIV-1 replication in human cells. We propose that PARP-1 may serve as a cellular target for RNA interference-mediated gene silencing to inhibit HIV-1 replication.

Inhibition of rat liver Ca2+, Mg2+-dependent endonuclease activity by nicotinamide adenine dinucleotide and poly(adenosine diphosphate ribose) synthetase

Summary Incubation of rat liver chromatin with NAD + resulted in an inhibition of the Ca 2+ , Mg 2+ -dependent endonuclease while the Mg 2+ -dependent endonuclease was not affected. To establish that the endonuclease was blocked directly by adenosine diphosphate ribosylation purified enzymes were used in the reaction mixture. The following ingredients were required in order to demonstrate the inhibitory effect: partially purified Ca 2+ , Mg 2+ -dependent endonuclease, purified poly(adenosine diphosphate ribose) synthetase, NAD + and DNA.

Poly(ADP-ribose) synthetase is phosphorylated by protein kinase C invitro

Poly (ADP-ribose) synthetase from bovine thymus was phosphorylated effectively by protein kinase C in vitro. The phosphorylation was dependent on the activators of this kinase, Ca2+ and phospholipid. The apparent Km for the synthetase was about 8 microM, which was lower than that for histone H1. Though the synthetase was a weak substrate for Ca2+/calmodulin-dependent protein kinase II, other protein kinases, cyclic AMP-dependent and cofactor-independent protein kinases did not phosphorylate the synthetase. Phosphorylation of the synthetase by protein kinase C resulted in appreciable inhibition of the synthetase activity.

Poly(ADP-ribosyl)ation of DNA polymerase βinvitro☆

DNA polymerase beta purified from bovine thymus is markedly inhibited when incubated in a reconstituted poly(ADP-ribosyl)ating reaction system. Analyses of the reaction product synthesized in this system by SDS-polyacrylamide gel electrophoresis and subsequent fluorography of the gel indicated that ADP-ribose is covalently attached to DNA polymerase beta molecule (Mr = 44,000).

Inhibition of ADP-ribosylation of histone by diadenosine 5′, 5‴-p1, p4-tetraphosphate

Abstract Diadenosine 5′, 5‴-p 1 , p 4 -tetraphosphate (Ap4A) strongly inhibited ADP-ribosylation reaction of histone by purified bovine thymus poly(ADP-ribose) polymerase. This compound showed a relatively weak inhibitory effect on Mg 2+ -dependent, enzyme-bound poly(ADP-ribose) synthesis. Among various adenine nucleotides tested, including several diadenosine nucleotides with varying phosphate chain length, Ap4A was the most effective inhibitor of the histone-modification reaction. Ap5A and Ap6A showed slightly lower inhibitory effect than Ap4A. Kinetic analysis of the inhibitor (Ap4A) with varying concentration of substrate (NAD + ) revealed that this compound is a “mixed type inhibitor”, with a Ki value of 5.1 μM.

Enzymic and nonenzymic mono ADP-ribosylation of proteins in skeletal muscle

The acceptors of endogenously catalyzed monoADP-ribosylation in the cell free extract from rat skeletal muscle was searched. The main acceptor proteins in particulate were electrophoretically 52, 80, 100, and greater than 200 kDa proteins in the presence of SDS, while that in cytosol were 36 and 39 kDa proteins. Although no ADP-ribosylation was observed in particulate when the substrate NAD+ was replaced by ADP-ribose, the same ADP-ribose adducts were also formed with higher degree in cytosol. These results indicate that an enzymic and nonenzymic monoADP-ribosylation occur separately in cytosol and particulate. One acceptor, 36 kDa protein, appears to be glyceraldehyde-3-phosphate dehydrogenase.

A method for determining oligo- and poly(ADP-ribosy)ated enzymes and proteins in vitro

A new method to determine oligo- and poly(ADP-ribosyl)ated enzymes and proteins in vitro has been developed. This method is based on the facts that in Mg2+-depleted condition automodification of poly(ADP-ribose)polymerase is minimized and exogenously added acceptor protein is oligo(ADP-ribosyl)ated predominantly, and in Mg2+-fortified conditions the exogenous acceptor can be poly(ADP-ribosyl)ated. When 13 proteins, including several enzymes, were subjected to this system, dimeric bovine seminal RNase and micrococcal nuclease were found to be oligo(ADP-ribosyl)ated under Mg2+-depleted conditions but their activity was unchanged. Under Mg2+-fortified conditions however, the RNase was deactivated concomitantly with its extensive poly(ADP-ribosyl)ation. When dimeric bovine seminal RNase was monomerized in advance by treatment with dithiothreitol and urea, the enzyme lost ADP-ribose-accepting ability in spite of a significant residual enzyme activity. As used here successfully, the Mg2+-depleted and Mg2+-fortified ADP-ribosylation and subsequent chromatographic analysis of various proteins and enzymes might be an useful method for proving their oligo- and poly(ADP-ribosyl)ation.

Release of DNA polymerase from rat liver chromatin on incubation with NAD

Rat liver nuclear chromatin possesses an enzymic activity which transfers the ADP-Rib at moiety of NAD to nucleoproteins to form a polymer [1 -3 ] . It was suggested that the formation of poly(ADP-Rib) might play a role in the regulation of DNA synthesis [4, 5] since its formation resulted in an inhibition of the template activity of isolated rat liver nuclei for DNA synthesis [4, 5]. This finding was confirmed by Nagao et al. [6]. They reported, however, that the endogenous DNA polymerase was inhibited rather than the template activity. In a previous communication we reported that the observed inhibition of DNA synthesis following incubation of isolated rat liver nuclei with NAD was associated with a block of the nuclear endonucleolytic activity [7]. In the present paper evidence will be presented to show that the observed inhibition of the endogenous DNA polymerase activity of rat liver chromatin following poly(ADP-Rib) formation was due to a liberation of the enzyme from chromatin into the incubation medium, paralleled with a loss of activity in the chromatin.