Asako Itaya

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.

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.

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).

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.

Stimulation of purified DNA polymerase α by various basic proteins which interact with activated DNA

Extensive purification of DNA polymerase alpha-primase resulted in a marked loss of the DNA polymerase alpha activity. This loss is due partly to the elimination of some basic proteins from the enzyme preparation since the activity of purified enzyme was stimulated 10- to 15-fold by the addition of various basic proteins, including all five classes of histones, protamine, poly-L-lysine, and poly-L-arginine, at a concentration of 2 micrograms/0.2 ml in the presence of 20 micrograms/0.2 ml of activated DNA. The optimum concentration of the basic proteins and the maximum activity attained at that concentration varied with varying concentrations of the template primer used, indicating that the observed stimulation is caused by an interaction between these basic proteins and activated DNA. The enzyme activity with an optimal concentration of activated DNA was markedly inhibited by the addition of denatured DNA. The suppressed enzyme activity could be restored by an appropriate concentration of histone H1. These results suggest that histone H1 and other basic proteins protect the enzyme from forming an abortive complex with single-stranded DNA or with a long stretch of the single-stranded part of activated DNA as single-stranded DNA-specific binding proteins do (M. Sapp, H. König, H. D. Riedel, A. Richter, and R. Knippers (1985) J. Biol. Chem. 260, 1550-1556). Spermine also showed a similar stimulatory effect. All acidic proteins tested were ineffective.

3-Aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, causes a rapid death of HL-60 cells cultured in serum-free medium

HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10μ/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serumdeprived growth condition.

Poly(ADP-Ribos)ylation of Nuclear Enzymes

Poly(ADP-ribose) polymerase catalyzes a sequential transfer of an ADP-ribose portion of NAD+ to various chromatin proteins [1] and to the polymerase itself (automodification [2]), forming a polymer of ADP-ribose, which is covalently bound to protein at one end [3]. Recent studies elucidated that two chromatin enzymes, Ca2+, Mg2+-dependent endonuclease [4, 5] and DNA topoisomerase [6, 7], were markedly inhibited as a result of poly(ADP-ribos)ylation of the enzyme proteins. RNA polymerase I [8] and DNA ligase II [9] also are suggested to be poly(ADP-ribos)ylated, although the latter enzyme seems to be activated after poly(ADP-ribos)ylation in vivo. Furthermore, bull seminal RNase [10, 29] and micrococcal nuclease [29] also have been shown to be the acceptors of ADP-ribose in the enzyme reaction in vitro. These results suggest a possibility that poly(ADP-ribose) polymerase randomly modifies many kinds of chromatin enzymes rather than it selecting a few kinds of specific enzymes as its targets. Thus, in order to study whether the modification reaction is specific only for the enzymes described above, we examined six nuclear enzymes, which are involved in metabolism or function of chromatin. After several unsuccessful trials using standard and modified conditions of the reconstituted ADP-ribosylating system, we found that all of these enzymes except DNA ligase I were markedly inhibited when the enzymes were incubated in an ADP-ribosylating reaction mixture containing a limited concentration of buffer (5 mM).

In Vitro Evidence for Poly(ADP-Ribosyl)ation of DNA Polymerase α-Primase and Phosphorylation of Poly(ADP-Ribose) Synthetase by Protein Kinase C

In a previous study (1–3), we found that terminal deoxyribonucleotidyl transferase (TdT), DNA polymerase α, DNA polymerase β, and DNA ligase II were markedly inhibited when incubated in a reconstituted poly(ADP-ribosyl)ating enzyme system. We have reported also the direct evidence for poly(ADP-ribosyl)ation of TdT (2) and DNA polymerase β (3). Based on these results, we proposed that a role of poly(ADP-ribose) synthetase in DNA repair is to cause an emergency halt of chromatin function at the damaged site to protect cells from abnormal metabolism in the chromatin (4).